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- Jarvius, Malin, et al.
(författare)
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In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method
- 2007
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 6:9, s. 1500-1509
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Tidskriftsartikel (refereegranskat)abstract
- Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor β (PDGFRβ) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRβ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRβ in porcine aortic endothelial cells transfected with the β-receptor, but not in cells transfected with the α-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRβ. We furthermore visualized tyrosine phosphorylated PDGFRβ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.
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| 2. |
- Weibrecht, Irene, et al.
(författare)
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Oxidation sensitivity of the catalytic cysteine of the protein-tyrosine phosphatases SHP-1 and SHP-2
- 2007
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Ingår i: Free Radical Biology & Medicine. - : Elsevier BV. - 0891-5849 .- 1873-4596. ; 43:1, s. 100-110
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Tidskriftsartikel (refereegranskat)abstract
- Reversible oxidation of the catalytic cysteine of protein-tyrosine phosphatases (PTPs) has emerged as a putative mechanism of activity regulation by physiological cell stimulation with growth factors, and by cell treatments with adverse agents such as UV irradiation. We compared SHP-1 and SHP-2, two structurally related cytoplasmic protein-tyrosine phosphatases with different cellular functions and cell-specific expression patterns, for their intrinsic susceptibility to oxidation by H(2)O(2). The extent of oxidation was monitored by detecting the modification of the PTP catalytic cysteine by three different methods, including a modified in-gel PTP assay, alkylation with a biotinylated iodoacetic acid derivative, and an antibody against oxidized PTPs. Dose-response curves for oxidation of the catalytic domains of SHP-1 and SHP-2 were similar. SHP-1 and -2 require relatively high H(2)O(2) concentrations for oxidation (half-maximal oxidation at 0.1-0.5 mM). For SHP-1, the SH2 domains had a significant protective function with respect to oxidation. In EOL-1 cells, SHP oxidation by exogenous H(2)O(2) in general and SHP-2 oxidation in particular was strongly diminished compared to HEK293 cells, at least partially related to a generally lower oxidant sensitivity of the EOL-1 cells. The data suggest that the differential cell functions of SHP-1 and SHP-2 are not related to differences in oxidation sensitivity. The modulating effects of SH2 domains for oxidation of these PTPs are in support of an enhanced oxidation susceptibility of activated SHPs.
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