Anna Maria Michela Di Stasi

Activation of src tyrosine kinases by peroxynitrite

In this study, we demonstrate that the phosphorylation activity of five tyrosine kinases of the src family from both human erythrocytes (lyn, hck and c‐fgr) and bovine synaptosomes (lyn and fyn) was stimulated by treatment with 30–250 μM peroxynitrite. This effect was not observed with syk, a non‐src family tyrosine kinase. Treatment of kinase immunoprecipitates with 0.01–10 μM peroxynitrite showed that the interaction of these enzymes with the oxidant also activated the src kinases. Higher concentrations of peroxynitrite inhibited the activity of all kinases, indicating enzyme inactivation. The addition of bicarbonate (1.3 mM CO2) did not modify the upregulation of src kinases but significantly protected the kinases against peroxynitrite‐mediated inhibition. Upregulation of src kinase activity by 1 μM peroxynitrite was 3.5–5‐fold in erythrocytes and 1.2–2‐fold in synaptosomes, but this could be the result, at least in part, of the higher basal level of src kinase activity in synaptosomes. Our results indicate that peroxynitrite can upregulate the tyrosine phosphorylation signal through the activation of src kinases.

Nitrotyrosine mimics phosphotyrosine binding to the SH2 domain of the src family tyrosine kinase lyn

The nitration of tyrosine residues in protein occurs through the action of reactive oxygen and nitrogen species and is considered a marker of oxidative stress under pathological conditions. The most active nitrating species so far identified is peroxynitrite, the product of the reaction between nitric oxide and superoxide anion. Previously, we have reported that in erythrocytes peroxynitrite irreversibly upregulates lyn, a tyrosine kinase of the src family. In this study we investigated the possible role of tyrosine nitration in the mechanism of lyn activation. We found that tyrosine containing peptides modelled either on the C‐terminal tail of src kinases or corresponding to the first 15 amino acids of human erythrocyte band 3 were able to activate lyn when the tyrosine was substituted with 3‐nitrotyrosine. The activity of nitrated peptides was shared with phosphorylated but not with unphosphorylated, chlorinated or scrambled peptides. Recombinant lyn src homology 2 (SH2) domain blocked the capacity of the band 3‐derived nitrotyrosine peptide to activate lyn and we demonstrated that this peptide specifically binds the SH2 domain of lyn. We propose that nitropeptides may activate src kinases through the displacement of the phosphotyrosine in the tail from its binding site in the SH2 domain. These observations suggest a new mechanism of peroxynitrite‐mediated signalling that may be correlated with the upregulation of tyrosine phosphorylation observed in several pathological conditions.

Neuronal fodrin proteolysis occurs independently of excitatory amino acid-induced neurotoxicity

In cultured cerebellar granule cells, the total amount of fodrin alpha subunit increased 3-fold between 0 and 10 days in vitro and fodrin mRNA increased 5-fold. The exposure of cerebellar neurons to NMDA induced the accumulation of a 150 kd proteolytic fragment of fodrin. The NMDA-induced breakdown of fodrin was time-, concentration-, and Ca2(+)-dependent and was inhibited by APV, Mg2+, or the calpain I inhibitor N-acetyl-Leu-Leu-norleucinal. Kainate caused fodrin proteolysis through indirect activation of NMDA receptors. Quisqualate was ineffective. The NMDA-induced degradation of fodrin occurred under conditions that did not cause degeneration of cultured cerebellar neurons. These results show that Ca2+/calpain I-dependent proteolysis of fodrin is selectively associated with NMDA receptor activation; however, fodrin proteolysis per se does not play a causal role in NMDA-induced toxicity in cerebellar granule cells.

Phosphorylation and nitration of tyrosine residues affect functional properties of Synaptophysin and Dynamin I, two proteins involved in exo-endocytosis of synaptic vesicles.

Phosphorylation and nitration of protein tyrosine residues are thought to play a role in signaling pathways at the nerve terminal and to affect functional properties of proteins involved in the synaptic vesicle (SV) exo-endocytotic cycle. We previously demonstrated that the tyrosine residues in the C-terminal domain of the SV protein Synaptophysin (SYP) are targets of peroxynitrite (PN). Here, we have characterized the association between SYP and c-src tyrosine kinase demonstrating that phosphorylation of Tyr(273) in the C-terminal domain of SYP is crucial in mediating SYP binding to and activation of c-src. SYP forms a complex with Dynamin I (DynI), a GTPase required for SV endocytosis, which may be regulated by tyrosine phosphorylation of SYP. We here report that, in rat brain synaptosomes treated with PN, the formation of SYP/DynI complex was impaired. Noteworthy, we found that DynI was also modified by PN. DynI tyrosine phosphorylation was down-regulated in a dose-dependent manner, while DynI tyrosine nitration increased. Using mass spectrometry analysis, we identified Tyr(354) as one nitration site in DynI. In addition, we tested DynI self-assembly and GTPase activity, which are enhanced by c-src-dependent tyrosine phosphorylation of DynI, and found that both were inhibited by PN. Our results suggest that the site-specific tyrosine residue modifications may modulate the association properties of SV proteins and serve as a regulator of DynI function via control of self-assembly, thus influencing the physiology of the exo-endocytotic cycle.

Peroxynitrite induces tyrosine residue modifications in synaptophysin C-terminal domain, affecting its interaction with src

Peroxynitrite is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. We have previously reported that treatment of rat brain synaptosomes with peroxynitrite induced post‐translational modifications in pre‐ and post‐synaptic proteins and stimulated soluble N‐ethylmaleimide sensitive fusion proteins attachment receptor complex formation and endogenous glutamate release. In this study we show that, following peroxynitrite treatment, the synaptic vesicle protein synaptophysin (SYP) can be both phosphorylated and nitrated in a dose‐dependent manner. We found that tyrosine‐phosphorylated, but not tyrosine‐nitrated, SYP bound to the src tyrosine kinase and enhanced its catalytic activity. These effects were mediated by direct and specific binding of the SYP cytoplasmic C‐terminal tail with the src homology 2 domain. Using mass spectrometry analysis, we mapped the SYP C‐terminal tail tyrosine residues modified by peroxynitrite and found one nitration site at Tyr250 and two phosphorylation sites at Tyr263 and Tyr273. We suggest that peroxynitrite‐mediated modifications of SYP may be relevant in modulating src signalling of synaptic terminal in pathophysiological conditions.

Early effects of high glucose in retinal tissue cultures Renin-Angiotensin system-dependent and -independent signaling.

The early effects of the diabetic milieu on retinal tissue and their relation to the Renin-Angiotensin system (RAS) activation are poorly known. Here we investigated RAS signaling in retinas explanted from adult rats exposed for 48 h to high glucose (HG), with or without the Angiotensin Converting Enzyme inhibitor enalaprilat, which blocks RAS. HG was observed to i) initiate a phosphotyrosine-dependent signaling cascade; ii) up-regulate Angiotensin(1) Receptor (AT(1)R); iii) activate src tyrosine kinase and increase phosphorylation of Pyk2, PLCgamma1 and ERK1/2; and iv) activate Akt and the transcription factor CREB. In the presence of enalaprilat, tyrosine phosphorylation signal and AT(1)R upregulation decreased and activation of PLCgamma1 and CREB reverted, showing their relation to RAS signaling. In line with Akt activation, no apoptosis or synapse degeneration was found. Müller glia was activated, but in a RAS-independent manner. Our results suggest that, in early phases of HG exposure, a pro-survival cell program may be induced in the retina.

Peroxynitrite-dependent upregulation of src kinases in red blood cells : Strategies to study the activation mechanisms

Several studies have demonstrated that treatment of cells with oxidants, and in particular with peroxynitrite, may cause the upregulation of tyrosine phosphorylation signaling. In erythrocytes, peroxynitrite induces tyrosine phosphorylation of the major intrinsic membrane protein, band 3. A closer look at the enzymes involved revealed that the effect of peroxynitrite was due to the inhibition of phosphotyrosine phosphatases and/or to the activation of src kinases. The activity of src kinases is modulated not only by phosphatases and other kinases but also through redox modification of cysteine residues: Peroxynitrite can, thus, affect src kinase activity by means of direct and indirect mechanisms. In this chapter, we describe the different pathways leading to src kinase activation and the experimental procedures that can be performed to reveal the activation mechanism. The aim is to provide a more general strategy adaptable to different cell types and different oxidants.

Cocaine-induced changes of synaptic transmission in the striatum are modulated by adenosine A2A receptors and involve the tyrosine phosphatase STEP.

The striatum is a brain area implicated in the pharmacological action of drugs of abuse. Adenosine A2A receptors (A2ARs) are highly expressed in the striatum and mediate, at least in part, cocaine-induced psychomotor effects in vivo. Here we studied the synaptic mechanisms implicated in the pharmacological action of cocaine in the striatum and investigated the influence of A2ARs. We found that synaptic transmission was depressed in corticostriatal slices after perfusion with cocaine (10 μM). This effect was reduced by the A2AR antagonist ZM241385 and almost abolished in striatal A2AR-knockout mice (mice lacking A2ARs in striatal neurons, stA2ARKO). The effect of cocaine on synaptic transmission was also prevented by the protein tyrosine phosphatases (PTPs) inhibitor sodium orthovanadate (Na3VO4). In synaptosomes prepared from striatal slices, we found that the activity of striatal-enriched protein tyrosine phosphatase (STEP) was upregulated by cocaine, prevented by ZM241385, and absent in synaptosomes from stA2ARKO. The role played by STEP in cocaine modulation of synaptic transmission was investigated in whole-cell voltage clamp recordings from medium spiny neurons of the striatum. We found that TAT-STEP, a peptide that renders STEP enzymatically inactive, prevented cocaine-induced reduction in AMPA- and NMDA-mediated excitatory post-synaptic currents, whereas the control peptide, TAT-myc, had no effect. These results demonstrate that striatal A2ARs modulate cocaine-induced synaptic depression in the striatum and highlight the potential role of PTPs and specifically STEP in the effects of cocaine.

Comparison of thermal properties of bovine spectrin and fodrin

Thermal properties of bovine brain fodrin have been studied by circular dichroism and electron spin resonance and compared to those of bovine erythrocyte spectrin. Protein unfolding was induced either by urea or by a combination of heat and urea. The denaturation profiles of the two proteins, as measured by the changes in ellipticity at 222 nm as a function of temperature, were very similar but fodrin denaturation occurred at both higher temperatures and higher urea concentrations. In the absence of urea the thermal transition of spectrin was centered at 51 degrees C and that of fodrin at 54.5 degrees C. Proteins were also labeled with a maleimide analog spin probe. Spin-labeled fodrin showed a thermal transition similar to that of spectrin but centered at 46 degrees C instead of 42 degrees C. These findings indicated a close structural similarity of the two proteins but a slightly higher conformational stability of fodrin to both heat and urea.