Nano-size uni-lamellar lipodisq improved in situ auto-phosphorylation analysis of E. coli tyrosine kinase using (19)F nuclear magnetic resonance.
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ABSTRACT:
SUBMITTER: Li D
PROVIDER: S-EPMC4348245 | biostudies-literature | 2015 Mar
REPOSITORIES: biostudies-literature
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Nano-size uni-lamellar lipodisq improved in situ auto-phosphorylation analysis of E. coli tyrosine kinase using (19)F nuclear magnetic resonance.
Li Dong D Li Juan J Zhuang Yonglong Y Zhang Longhua L Xiong Ying Y Shi Pan P Tian Changlin C
Protein & cell 20150301 3
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Project description:BackgroundTyrosine phosphorylation (pTyr) is an important cancer relevant posttranslational modification since it regulates protein activity and cellular localization. By controlling cell growth and differentiation it plays an important role in tumor development. This paper describes a novel approach for detection and visualization of a panel of pTyr proteins in tumors using in situ proximity ligation assay.MethodsK562 leukemia cells were treated with tyrosine kinase and/or phosphatase inhibitors to induce differences in pTyr levels and mimic cells with different malignant properties. Cells were then probed with one antibody against the pTyr modification and another probe against the detected protein, resulting in a detectable fluorescent signal once the probes were in proximity.ResultsTotal and protein specific pTyr levels on ABL, SHC, ERK2 and PI3K proteins were detected and samples of control and treated cells were distinguished at the pTyr level using this novel approach. Promising results were also detected for formalin fixed and paraffin embedded cells in the micro array format.ConclusionsThis application of in situ proximity ligation assay is valuable in order to study the pTyr modification of a panel of proteins in large data sets to validate mass spectrometric data and to be combined with tissue microarrays. The approach offers new opportunities to reveal the pTyr signatures in cells of different malignant properties that can be used as biomarker of disease in the future.
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Project description:Focal adhesion kinase (FAK) is an intensely studied non-receptor tyrosine kinase with roles in cancer and other common human diseases. Despite the large interest in FAK, the in vivo contribution of FAK auto-phosphorylation site tyrosine (Y) 397 to FAK function is incompletely understood. To study FAK Y397 in vivo we analyzed mice with 'non-phosphorylatable' Y-to-phenylalanine (F) and 'phospho-mimicking' Y-to-glutamate (E) mutations in the germline. We found that FAK Y397F mice die early during embryogenesis with abnormal angiogenesis like FAK kinase-dead mice. When Y397 is mutated to a glutamate mice survive beyond mid-gestation like mice where Y397 is lost by deletion of FAK exon 15. In culture, defects in proliferation, invasion and gene expression were more severe with the FAK Y397F than with the FAK Y397E mutation despite the inability of FAK Y397E to bind SRC. Conditional expression of FAK Y397F or Y397E in unchallenged avascular epidermis, however, resulted in no appreciable phenotype. We conclude that FAK Y397 is required for the highly dynamic tissue remodeling during development but dispensable for normal homeostasis of avascular epidermis. In contrast to the Y397F mutation, FAK Y397E retains sufficient biological activity to allow for development beyond mid-gestation.
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Project description:The ability of mixtures of 1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the amphiphilic diblock copolymers poly (ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) to stabilize uni-lamellar nano-vesicles is reported. Small angle neutron scattering (SANS) is used to define their size distribution and bilayer structure and resolve the copresence of aggregates and clusters in solution. The vesicles have a broad size distribution which is compatible with bilayer membranes of relatively low bending stiffness. Their mean diameter increases moderately with temperature and their number density and mass is higher in the case of the diblock copolymer with the larger hydrophobic block. Bayesian analysis is performed in order to justify the use of the particular SANS fitting model and confirm the reliability of the extracted parameters. This study shows that amphiphilic block copolymers can be effectively used to prepare mixed lipid-block copolymer vesicles with controlled lamellarity and a significant potential as nanocarriers for drug delivery.
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Project description:Syk is a cytoplasmic tyrosine kinase that is activated after recruitment to immune receptors, triggering the phopshorylation of downstream targets. The kinase activity of Syk is controlled by an auto-inhibited conformation consisting of a regulatory region that contains two N-terminal Src homology 2 (SH2) domains inhibiting the catalytic activity of the kinase domain located at the C-terminus. The atomic structure of the related Zap-70 kinase and an electron microscopy (EM) model of Syk have revealed the structural mechanism of this auto-inhibition based on the formation of a compact conformation sustained by interactions between the regulatory and catalytic domains. On the other hand, the structural basis of Syk activation is not fully understood due to the lack of a 3D structure of full-length Syk in an active conformation. Here, we have used single-particle electron microscopy to analyse the conformational changes taking place in an activated form of Syk induced by auto-phosphorylation. The conformation of phosphorylated Syk is reminiscent of the compact structure of the inhibited protein but significant conformational changes are observed in the regulatory region. These rearrangements could be sufficient to disrupt the inhibitory interactions, contributing to Syk activation. These results suggest that the regulation of the activation of Syk might be modulated by subtle changes in the positioning of the regulatory domains rather than a full opening mechanism as proposed for the Src kinases.
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Project description:BackgroundIntermolecular autophosphorylation at Tyr416 is a conserved mechanism of activation among the members of the Src family of nonreceptor tyrosine kinases. Like several other tyrosine kinases, Src can catalyze the thiophosphorylation of peptide and protein substrates using ATPγS as a thiophosphodonor, although the efficiency of the reaction is low.ResultsHere, we have characterized the ability of Src to auto-thiophosphorylate. Auto-thiophosphorylation of Src at Tyr416 in the activation loop proceeds efficiently in the presence of Ni(2+), resulting in kinase activation. Other tyrosine kinases (Ack1, Hck, and IGF1 receptor) also auto-thiophosphorylate in the presence of Ni(2+). Tyr416-thiophosphorylated Src is resistant to dephosphorylation by PTP1B phosphatase.ConclusionsSrc and other tyrosine kinases catalyze auto-thiophosphorylation in the presence of Ni(2+). Thiophosphorylation of Src occurs at Tyr416 in the activation loop, and results in enhanced kinase activity. Tyr416-thiophosphorylated Src could serve as a stable, persistently-activated mimic of Src.
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Project description:Concentration gradients provide spatial information for tissue patterning and cell organization, and their robustness under natural fluctuations is an evolutionary advantage. In rod-shaped Schizosaccharomyces pombe cells, the DYRK-family kinase Pom1 gradients control cell division timing and placement. Upon dephosphorylation by a Tea4-phosphatase complex, Pom1 associates with the plasma membrane at cell poles, where it diffuses and detaches upon auto-phosphorylation. Here, we demonstrate that Pom1 auto-phosphorylates intermolecularly, both in vitro and in vivo, which confers robustness to the gradient. Quantitative imaging reveals this robustness through two system's properties: The Pom1 gradient amplitude is inversely correlated with its decay length and is buffered against fluctuations in Tea4 levels. A theoretical model of Pom1 gradient formation through intermolecular auto-phosphorylation predicts both properties qualitatively and quantitatively. This provides a telling example where gradient robustness through super-linear decay, a principle hypothesized a decade ago, is achieved through autocatalysis. Concentration-dependent autocatalysis may be a widely used simple feedback to buffer biological activities.
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