title: Phosphorylation and nitration of tyrosine residues affect functional properties of Synaptophysin and Dynamin I, two proteins involved in exo-endocytosis of synaptic vesicles.
abstract: 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.
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy. cinzia.mallozzi@iss.it
authors.forename: Cinzia
authors.initials: C
authors.lastname: Mallozzi
authors.forename: Carmen
authors.initials: C
authors.lastname: D'Amore
authors.forename: Serena
authors.initials: S
authors.lastname: Camerini
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Macchia
authors.forename: Marco
authors.initials: M
authors.lastname: Crescenzi
authors.forename: Tamara Corinna
authors.initials: TC
authors.lastname: Petrucci
authors.forename: Anna Maria Michela
authors.initials: AM
authors.lastname: Di Stasi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D034284:Dynamin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D004705:Endocytosis / Q000235:genetics / Q000502:physiology; D005089:Exocytosis / Q000235:genetics / Q000502:physiology; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009566:Nitrates / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000502:physiology; D016708:Synaptophysin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism / Q000502:physiology
chemical_list: D009566:Nitrates; D016708:Synaptophysin; C569016:Syp protein, rat; D014443:Tyrosine; D011494:Protein Kinases; D034284:Dynamin I