scholarly journals Modification by covalent reaction or oxidation of cysteine residues in the tandem-SH2 domains of ZAP-70 and Syk can block phosphopeptide binding

2014 ◽  
Vol 465 (1) ◽  
pp. 149-161 ◽  
Author(s):  
Patrick R. Visperas ◽  
Jonathan A. Winger ◽  
Timothy M. Horton ◽  
Neel H. Shah ◽  
Diane J. Aum ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Immune Cell ◽  
Cell Signalling ◽  
Cysteine Residues ◽  
Spleen Tyrosine Kinase ◽  
Sh2 Domains ◽  
Src Homology

H2O2 and thiol-reactive inhibitors can attenuate the binding of the tandem-SH2 (Src-homology domain 2) domains of ζ-chain associated protein of 70 kDa (ZAP-70) and spleen tyrosine kinase (Syk) to doubly phosphorylated ITAM. This suggests a novel mechanism whereby oxidation of cysteine residues in these kinases could regulate immune cell signalling.

scholarly journals Structure of the interleukin-2 tyrosine kinase Src homology 2 domain; comparison between X-ray and NMR-derived structures

2012 ◽  
Vol 68 (2) ◽  
pp. 145-153 ◽  
Author(s):  
Raji E. Joseph ◽  
Nathaniel D. Ginder ◽  
Julie A. Hoy ◽  
Jay C. Nix ◽  
D. Bruce Fulton ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nmr Spectroscopy ◽  
Tyrosine Kinase ◽  
Interleukin 2 ◽  
Crystal Form ◽  
X Ray ◽  
Proline Isomerization ◽  
Sh2 Domains ◽  
X Ray Crystallography ◽  
Src Homology

The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy,cis–trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and thecisform of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts thecisconformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolylcis–transisomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the β-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerizationversusdomain-swapped dimerization, respectively.

scholarly journals Integrin cytoplasmic domain and pITAM compete for spleen tyrosine kinase binding

2019 ◽  
Author(s):  
Lina Antenucci ◽  
Maarit Hellman ◽  
Vesa P. Hytönen ◽  
Perttu Permi ◽  
Jari Ylänne
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Binding Site ◽  
Conformational Changes ◽  
Cytoplasmic Domain ◽  
Sh2 Domain ◽  
Regulatory Domain ◽  
Spleen Tyrosine Kinase ◽  
Adhesion Receptors ◽  
Sh2 Domains

ABSTRACTIn hematopoietic tissues cell-cell communication involves immunoreceptors and specialized cell adhesion receptors that both mediate intracellular signals. Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase involved in the downstream signaling of both immunoreceptors tyrosine activation motif (ITAM) receptors and integrin family cell adhesion receptors. Both phosphorylated ITAM (pITAM) and integrins bind to the regulatory domain of Syk composed of two Src homology 2 (SH2) domains. The interaction with pITAM is mediated by binding of a specific phosphotyrosine to each of the SH2 domains, leading to conformational changes and Syk kinase activation. Integrins bind to the interdomain A segment between the two SH2 domains and to the N-terminal SH2 domain, but the detailed binding site is not known. In order to map the binding site, we performed NMR titration experiments. We found that integrin cytoplasmic domain peptide induced chemical shift changes near the IA segment and at the phosphotyrosine binding site of the N-terminal SH2 domain of Syk. These changes were distinct, but partially overlapping with those induced by pITAM peptide. We were also able to show that pITAM peptide inhibited integrin binding to Syk regulatory domain. These results suggest that ITAM receptors and integrins cannot bind simultaneously to Syk, but provide two distinct routes for Syk activation.

scholarly journals Spleen Tyrosine Kinase Is a Critical Regulator of Neutrophil Responses to Candida Species

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Paige E. Negoro ◽  
Shuying Xu ◽  
Zeina Dagher ◽  
Alex Hopke ◽  
Jennifer L. Reedy ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Essential Role ◽  
Immune Cell ◽  
Fungal Infections ◽  
Lectin Binding ◽  
Invasive Fungal Infections ◽  
Solid Organ ◽  
Spleen Tyrosine Kinase ◽  
Cellular Activation ◽  
Content Type

ABSTRACT Invasive fungal infections constitute a lethal threat, with patient mortality as high as 90%. The incidence of invasive fungal infections is increasing, especially in the setting of patients receiving immunomodulatory agents, chemotherapy, or immunosuppressive medications following solid-organ or bone marrow transplantation. In addition, inhibitors of spleen tyrosine kinase (Syk) have been recently developed for the treatment of patients with refractory autoimmune and hematologic indications. Neutrophils are the initial innate cellular responders to many types of pathogens, including invasive fungi. A central process governing neutrophil recognition of fungi is through lectin binding receptors, many of which rely on Syk for cellular activation. We previously demonstrated that Syk activation is essential for cellular activation, phagosomal maturation, and elimination of phagocytosed fungal pathogens in macrophages. Here, we used combined genetic and chemical inhibitor approaches to evaluate the importance of Syk in the response of neutrophils to Candida species. We took advantage of a Cas9-expressing neutrophil progenitor cell line to generate isogenic wild-type and Syk-deficient neutrophils. Syk-deficient neutrophils are unable to control the human pathogens Candida albicans, Candida glabrata, and Candida auris. Neutrophil responses to Candida species, including the production of reactive oxygen species and of cytokines such as tumor necrosis factor alpha (TNF-α), the formation of neutrophil extracellular traps (NETs), phagocytosis, and neutrophil swarming, appear to be critically dependent on Syk. These results demonstrate an essential role for Syk in neutrophil responses to Candida species and raise concern for increased fungal infections with the development of Syk-modulating therapeutics. IMPORTANCE Neutrophils are recognized to represent significant immune cell mediators for the clearance and elimination of the human-pathogenic fungal pathogen Candida. The sensing of fungi by innate cells is performed, in part, through lectin receptor recognition of cell wall components and downstream cellular activation by signaling components, including spleen tyrosine kinase (Syk). While the essential role of Syk in macrophages and dendritic cells is clear, there remains uncertainty with respect to its contribution in neutrophils. In this study, we demonstrated that Syk is critical for multiple cellular functions in neutrophils responding to major human-pathogenic Candida species. These data not only demonstrate the vital nature of Syk with respect to the control of fungi by neutrophils but also warn of the potential infectious complications arising from the recent clinical development of novel Syk inhibitors for hematologic and autoimmune disorders.

Phosphatidylinositol 3-kinase–dependent translocation of phospholipase Cγ2 in mouse megakaryocytes is independent of Bruton tyrosine kinase translocation

Blood ◽  
2001 ◽  
Vol 97 (3) ◽  
pp. 678-684 ◽  
Author(s):  
Régis Bobe ◽  
Jonathan I. Wilde ◽  
Petra Maschberger ◽  
Kanamarlapudi Venkateswarlu ◽  
Peter J. Cullen ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Ph Domain ◽  
Sh2 Domains ◽  
Glycoprotein Vi ◽  
Bruton Tyrosine Kinase ◽  
Src Homology ◽  
Epidermal Growth ◽  
Green Fluorescent

Abstract Activation of the collagen receptor glycoprotein VI (GPVI) by a collagen-related peptide (CRP) induces stimulation of platelets and megakaryocytes through the phosphatidylinositol (PI) 3-kinase–dependent pathway leading to activation of Bruton tyrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2). Here, we present evidence that both proteins undergo PI 3-kinase–dependent translocation to the plasma membrane on CRP stimulation that is markedly inhibited by wortmannin and LY294002. Translocation of PLCγ2 but not Btk is also seen in megakaryocytes from X-linked immunodeficiency mice, which have a mutation that reduces the affinity of the pleckstrin homology (PH) domain of Btk for PI 3,4,5-trisphosphate (PI 3,4,5-P3). Activation of PC12 cells by epidermal growth factor (EGF) results in increased PI 3-kinase activity and high PI 3,4,5-P3 levels that trigger translocation of the green fluorescent protein (GFP)–labeled PH of Btk, but not the GFP-labeled PH and tandem Src homology 2 (SH2) domains of PLCγ2. In contrast to the results with CRP, the G protein–coupled receptor agonist thrombin stimulates PI 3-kinase–independent translocation of Btk but not PLCγ2. In conclusion, these results demonstrate that in mouse megakaryocytes, CRP leads to PI 3-kinase–dependent translocation of PLCγ2 and Btk that are independent of one another, whereas thrombin only induces translocation of Btk through a pathway that is independent of PI 3-kinase activity.

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