Comparison of different strategies to select aptamers against a transmembrane protein target

Binding of aptamers is dependent on their target conformation, which in turn is conditioned by the target's environment. Therefore, selection of aptamers against the active forms of membrane proteins could require their correct membrane insertion in order to maintain their native conformation. Here, we compare different SELEX strategies to identify aptamers against the mutated form of the membrane receptor tyrosine kinase RET(C634Y). (1) selections S1 and S2 against living cells transformed to express the protein yielded a minority of RET-targeted aptamers while the bulk of aptamers recognized more abundant membrane proteins, suggesting that a high level of expression of the target protein is crucial to allow the isolation of aptamers at cell surface; (2) selection S3 against the purified extracellular moiety of RET yielded aptamers unable to recognize RET expressed at the cell membrane; (3) crossover selections S4 and S5 alternating cells and recombinant RET enhanced the enrichment of the aptamers directed against RET; however, these aptamers displayed a weaker affinity for Ret than those obtained with S1 and S2. In our case, using transformed cell lines as the partitioning matrix during SELEX appears to be essential in order to obtain aptamers able to recognize the RET receptor tyrosine kinase in its physiologic environment.     

Ultraviolet light induces redox reaction-mediated dimerization and superactivation of oncogenic Ret tyrosine kinases

The c-RET proto-oncogene encodes a receptor-type tyrosine kinase, and its mutations in the germ line are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B). Ret kinases are constitutively activated as a result of MEN2A mutations (Ret-MEN2A) or MEN2B mutations (Ret-MEN2B). Here we demonstrate that UV light (UV) irradiation induces superactivation of the constitutively activated Ret-MEN2A and Ret-MEN2B as well as activation of c-Ret. Before UV irradiation, small percentages of c-Ret (3–4%) and Ret-MEN2B (1–2%) and large percentages of Ret-MEN2A (30–40%) were dimerized through disulfide bonds. These dimerized Ret proteins were preferentially autophosphorylated, suggesting a close relation between up-regulated kinase activity and disulfide bond–mediated dimerization of Ret proteins. We found that UV irradiation promotes the disulfide bond–mediated dimerization of the Ret proteins, in close association with activation and superactivation of Ret kinases. UV irradiation also induced dimerization and activation of the extracellular domain–deleted mutant Ret (Ret-PTC-1). Interestingly, the levels of basic kinase activity and dimerization of Ret-PTC-1–C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-PTC-1 was replaced by alanine, were low and were not increased by UV irradiation. These results suggest that Ret-PTC-1 cysteine 376 is one of possibly multiple critical target amino acids of UV for Ret kinase activation. Overexpression of Cu/Zn superoxide dismutase in cells as a result of gene transfection prevented both the UV-mediated promotion of dimerization and the superactivation of Ret-MEN2A kinase. These results suggest that the UV-induced free radicals in cells attack intracellular domains of Ret to dimerize the kinase proteins for superactivation.     

A redox‐linked novel pathway for arsenic‐mediated RET tyrosine kinase activation

We examined the biochemical effects of arsenic on the activities of RET proto‐oncogene (c‐RET protein tyrosine kinases) and RET oncogene (RET‐MEN2A and RET‐PTC1 protein tyrosine kinases) products. Arsenic activated c‐RET kinase with promotion of disulfide bond‐mediated dimerization of c‐RET protein. Arsenic further activated RET‐MEN2A kinase, which was already 3‐ to 10‐fold augmented by genetic mutation compared with c‐RET kinase activity, with promotion of disulfide bond‐mediated dimerization of RET‐MEN2A protein (superactivation). Arsenic also increased extracellular domain‐deleted RET‐PTC1 kinase activity with promotion of disulfide bond‐mediated dimerization of RET‐PTC1 protein. Arsenic increased RET‐PTC1 kinase activity with cysteine 365 (C365) replaced by alanine with promotion of dimer formation but not with cysteine 376 (C376) replaced by alanine. Our results suggest that arsenic‐mediated regulation of RET kinase activity is dependent on conformational change of RET protein through modulation of a special cysteine sited at the intracellular domain in RET protein (relevant cysteine of C376 in RET‐PTC1 protein). Moreover, arsenic enhanced the activity of immunoprecipitated RET protein with increase in thiol‐dependent dimer formation. As arsenic (14.2 µM) was detected in the cells cultured with arsenic (100 µM), direct association between arsenic and RET in the cells might modulate dimer formation. Thus, we demonstrated a novel redox‐linked mechanism of activation of arsenic‐mediated RET proto‐oncogene and oncogene products. J. Cell. Biochem. 110: 399–407, 2010. © 2010 Wiley‐Liss, Inc.     

Study of the fast-reacting cysteines in phosphoglycerate kinase using chemical modification and site-directed mutagenesis

Horse muscle phosphoglycerate kinase, like other mammalian phosphoglycerate kinases, contains seven cysteine residues of which two react rapidly with 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) following second-order kinetics (k = 640 M-1.s-1). Selective cyanylation of the fast-reacting cysteines, followed by chemical cleavage and subsequent sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of the resulting polypeptides, suggested that these cysteines are at positions 378 and 379. Cysteine residues were introduced into yeast phosphoglycerate kinase by site-directed mutagenesis. Mutant enzymes, each containing only one cysteine residue at position 364, 376, or 377, were constructed from a mutant devoid of cysteine (Cys97----Ala). In the last two mutants, the cysteines were at positions corresponding to Cys378 and Cys379, respectively, in the horse muscle enzyme. The chemical reactivity of the cysteine groups in these latter two yeast mutant enzymes was similar to that of the fast-reacting cysteines in the horse muscle enzyme. Furthermore, they were similarly modified upon substrate binding. All these data demonstrate unambiguously that the fast-reacting cysteines in the horse muscle enzyme are Cys378 and Cys379.     

RET/PTC (rearranged in transformation/papillary thyroid carcinomas) tyrosine kinase phosphorylates and activates phosphoinositide-dependent kinase 1 (PDK1): an alternative phosphatidylinositol 3-kinase-independent pathway to activate PDK1

Thyroid cancers are a leading cause of death due to endocrine malignancies. RET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although the oncogenic potential of RET/PTC is related to intrinsic tyrosine kinase activity, the substrates for this enzyme are yet to be identified. In this report, we show that phosphoinositide-dependent kinase 1 (PDK1), a pivotal serine/threonine kinase in growth factor-signaling pathways, is a target of RET/PTC. RET/PTC and PDK1 colocalize in the cytoplasm. RET/PTC phosphorylates a specific tyrosine (Y9) residue located in the N-terminal region of PDK1. Y9 phosphorylation of PDK1 by RET/PTC requires an intact catalytic kinase domain. The short (iso 9) and long forms (iso 51) of the RET/PTC kinases (RET/PTC1 and RET/PTC3) induce Y9 phosphorylation of PDK1. Moreover, Y9 phosphorylation of PDK1 by RET/PTC does not require phosphatidylinositol 3-kinase or Src activity. RET/PTC-induced phosphorylation of the Y9 residue results in increased PDK1 activity, decrease of cellular p53 levels, and repression of p53-dependent transactivation. In conclusion, RET/PTC-induced tyrosine phosphorylation of PDK1 may be one of the mechanisms by which it acts as an oncogenic tyrosine kinase in thyroid carcinogenesis.     

Induction of heat shock proteins (HSPs) by sodium arsenite in cultured astrocytes and reduction of hydrogen peroxide-induced cell death

Induction of heat shock proteins (HSPs) protects cells from oxidative injury. Here Hsp72, Hsp27 and heme oxygenase-1 (HO-1) were induced in cultured rat astrocytes, and protection against oxidative stress was investigated. Astrocytes were treated with sodium arsenite (20-50 micro m) for 1 h, which was non-toxic to cells, 24 h later they were exposed to 400 micro m H2O2 for 1 h, and cell death was evaluated at different time points. Arsenite triggered strong induction of HSPs, which was prevented by 1 micro g/mL cycloheximide (CXH). H2O2 caused cell loss and increased cell death with features of apoptosis, i.e. TdT-mediated dUTP nick-end labelling (TUNEL) reaction and caspase-3 activation. These features were abrogated by pre-treatment with arsenite, which prevented cell loss and significantly reduced the number of dead cells. The protective effect of arsenite was not detected in the presence of CHX. Pre-treatment with arsenite increased protein kinase B (Akt) and extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation after H2O2. However, while Akt phosphorylation was prevented by CHX, Erk1/2 phosphorylation was further enhanced by CHX. The results show that transient arsenite pre-treatment induces Hsp72, HO-1 and, to a lesser extent, Hsp27; it reduces H2O2-induced astrocyte death; and it causes selective activation of Akt following H2O2. It is suggested that HSP expression at the time of H2O2 exposure protects astrocytes from oxidative injury and apoptotic cell death by means of pro-survival Akt.     

Critical cysteines in Akt1 regulate its activity and proteasomal degradation: implications for neurodegenerative diseases

Impaired Akt1 signaling is observed in neurodegenerative diseases, including Parkinson׳s disease (PD). In PD models oxidative modification of Akt1 leads to its dephosphorylation and consequent loss of its kinase activity. To explore the underlying mechanism we exposed Neuro2A cells to cadmium, a pan inhibitor of protein thiol disulfide oxidoreductases, including glutaredoxin 1 (Grx1), or downregulated Grx1, which led to dephosphorylation of Akt1, loss of its kinase activity, and also decreased Akt1 protein levels. Mutation of cysteines to serines at 296 and 310 in Akt1 did not affect its basal kinase activity but abolished cadmium- and Grx1 downregulation-induced reduction in Akt1 kinase activity, indicating their critical role in redox modulation of Akt1 function and turnover. Cadmium-induced decrease in phosphorylated Akt1 correlated with increased association of wild-type (WT) Akt1 with PP2A, which was absent in the C296–310S Akt1 mutant and was also abolished by N-acetylcysteine treatment. Further, increased proteasomal degradation of Akt1 by cadmium was not seen in the C296–310S Akt1 mutant, indicating that oxidation of cysteine residues facilitates degradation of WT Akt1. Moreover, preventing oxidative modification of Akt1 cysteines 296 and 310 by mutating them to serines increased the cell survival effects of Akt1. Thus, in neurodegenerative states such as PD, maintaining the thiol status of cysteines 296 and 310 in Akt1 would be critical for Akt1 kinase activity and for preventing its degradation by proteasomes. Preventing downregulation of Akt signaling not only has long-range consequences for cell survival but could also affect the multiple roles that Akt plays, including in the Akt–mTOR signaling cascade.     

Molecular modeling of the extracellular domain of the RET receptor tyrosine kinase reveals multiple cadherin-like domains and a calcium-binding site.

Using bioinformatic tools, mutagenesis, and binding studies, we have investigated the structural organization of the extracellular region of the RET receptor tyrosine kinase, a functional receptor for glial cell line-derived neurotrophic factor (GDNF). Multiple sequence alignments of seven vertebrate sequences and one invertebrate RET sequence delineated four distinct N-terminal domains, each of about 110 residues, containing many of the consensus motifs of the cadherin fold. Based on these alignments and the crystal structures of epithelial and neural cadherins, we have generated molecular models of each of the four cadherin-like domains in the extracellular region of human RET. The modeled structures represent realistic models from both energetic and geometrical points of view and are consistent with previous observations gathered from biochemical analyses of the effects of Hirschsprung's disease mutations affecting the folding and stability of the RET molecule, as well as our own site-directed mutagenesis studies of RET cadherin-like domain 1. We have also investigated the role of Ca(2+) in ligand binding by RET and found that Ca(2+) ions are required for RET binding to GDNF but not for GDNF binding to the GFRalpha1 co-receptor. In agreement with these results, RET, but not GFRalpha1, was found to bind Ca(2+) directly. Our results indicate that the overall architecture of the extracellular region of RET is more closely related to cadherins than previously thought. The models of the cadherin-like domains of human RET represent valuable tools with which to guide future site-directed mutagenesis studies aimed at identifying residues involved in ligand binding and receptor activation.     

1,4‐butanediyl‐bismethanethiosulfonate (BMTS) induces apoptosis through reactive oxygen species‐mediated mechanism

Although methane sulfonate compounds are widely used for the protein modification for their selectivity of thiol groups in proteins, their intracellular signaling events have not yet been clearly documented. This study demonstrated the methane sulfonate chemical 1,4‐butanediyl‐bismethanethiosulfonate (BMTS)‐induced cascades of signals that ultimately led to apoptosis of Jurkat cells. BMTS induced apoptosis through fragmentation of DNA, activation of caspase‐9 and caspase‐3, and downregulation of Bcl‐2 protein with reduction of mitochondrial membrane potential. Moreover, BMTS intensely and transiently induced intracellular reactive oxygen species (ROS) production and ROS produced by BMTS was mediated through mitochondria. We also found that a reducing agent dithiothreitol (DTT) and an anti‐oxidant N‐acetyl cysteine (NAC) inhibited BMTS‐mediated caspase‐9 and ‐3 activation, ROS production and induction of Annexin V/propidium iodide double positive cells, suggesting the involvement of ROS in the apoptosis process. Therefore, this study further extends our understanding on the basic mechanism of redox‐linked apoptosis induced by sulfhydryl‐reactive chemicals. J. Cell. Biochem. 108: 1059–1065, 2009. © 2009 Wiley‐Liss, Inc.     

Crosstalk between VEGF-A/VEGFR2 and GDNF/RET signaling pathways

Vascular endothelial growth factor (VEGF-A) plays multiple roles in kidney development: stimulates cell proliferation, survival, tubulogenesis, and branching morphogenesis. However, the mechanism that mediates VEGF-A induced ureteric bud branching is unclear. Glial-derived neurotrophic factor (GDNF) signaling through tyrosine kinase c-RET is the major regulator of ureteric bud branching. Here we examined whether VEGF-A regulates RET signaling. We determined that ureteric bud-derived cells express the main VEGF-A signaling receptor, VEGFR2 and RET, by RT-PCR, immunoblotting, and immunocytochemistry. We show that the VEGF-A isoform VEGF(165) induces RET-tyr(1062) phosphorylation in addition to VEGFR2 autophosphorylation, that VEGF(165) and GDNF have additive effects on RET-tyr(1062) phosphorylation, and that VEGFR2 and RET co-immunoprecipitate. Functionally, VEGF(165) induces ureteric bud cell proliferation and branching morphogenesis. Similarly, in embryonic kidney explants VEGF(165) induces RET-tyr(1062) phosphorylation and upregulates GDNF. These findings provide evidence for a novel cooperative interaction between VEGFR2 and RET that mediates VEGF-A functions in ureteric bud cells.