Binding of Protein Kinase Inhibitors to Synapsin I Inferred from Pair-Wise Binding Site Similarity Measurements

Predicting off-targets by computational methods is getting increasing importance in early drug discovery stages. We herewith present a computational method based on binding site three-dimensional comparisons, which prompted us to investigate the cross-reaction of protein kinase inhibitors with synapsin I, an ATP-binding protein regulating neurotransmitter release in the synapse. Systematic pair-wise comparison of the staurosporine-binding site of the proto-oncogene Pim-1 kinase with 6,412 druggable protein-ligand binding sites suggested that the ATP-binding site of synapsin I may recognize the pan-kinase inhibitor staurosporine. Biochemical validation of this hypothesis was realized by competition experiments of staurosporine with ATP-γ³⁵S for binding to synapsin I. Staurosporine, as well as three other inhibitors of protein kinases (cdk2, Pim-1 and casein kinase type 2), effectively bound to synapsin I with nanomolar affinities and promoted synapsin-induced F-actin bundling. The selective Pim-1 kinase inhibitor quercetagetin was shown to be the most potent synapsin I binder (IC50  = 0.15 µM), in agreement with the predicted binding site similarities between synapsin I and various protein kinases. Other protein kinase inhibitors (protein kinase A and chk1 inhibitor), kinase inhibitors (diacylglycerolkinase inhibitor) and various other ATP-competitors (DNA topoisomerase II and HSP-90α inhibitors) did not bind to synapsin I, as predicted from a lower similarity of their respective ATP-binding sites to that of synapsin I. The present data suggest that the observed downregulation of neurotransmitter release by some but not all protein kinase inhibitors may also be contributed by a direct binding to synapsin I and phosphorylation-independent perturbation of synapsin I function. More generally, the data also demonstrate that cross-reactivity with various targets may be detected by systematic pair-wise similarity measurement of ligand-annotated binding sites.     

Synapsin phosphorylation by SRC tyrosine kinase enhances SRC activity in synaptic vesicles

Synapsins are synaptic vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release. Synapsin I is the major binding protein for the SH3 domain of the kinase c-Src in synaptic vesicles. Its binding leads to stimulation of synaptic vesicle-associated c-Src activity. We investigated the mechanism and role of Src activation by synapsins on synaptic vesicles. We found that synapsin is tyrosine phosphorylated by c-Src in vitro and on intact synaptic vesicles independently of its phosphorylation state on serine. Mass spectrometry revealed a single major phosphorylation site at Tyr(301), which is highly conserved in all synapsin isoforms and orthologues. Synapsin tyrosine phosphorylation triggered its binding to the SH2 domains of Src or Fyn. However, synapsin selectively activated and was phosphorylated by Src, consistent with the specific enrichment of c-Src in synaptic vesicles over Fyn or n-Src. The activity of Src on synaptic vesicles was controlled by the amount of vesicle-associated synapsin, which is in turn dependent on synapsin serine phosphorylation. Synaptic vesicles depleted of synapsin in vitro or derived from synapsin null mice exhibited greatly reduced Src activity and tyrosine phosphorylation of other synaptic vesicle proteins. Disruption of the Src-synapsin interaction by internalization of either the Src SH3 or SH2 domains into synaptosomes decreased synapsin tyrosine phosphorylation and concomitantly increased neurotransmitter release in response to Ca(2+)-ionophores. We conclude that synapsin is an endogenous substrate and activator of synaptic vesicle-associated c-Src and that regulation of Src activity on synaptic vesicles participates in the regulation of neurotransmitter release by synapsin.     

Kidins220/ARMS is a novel modulator of short-term synaptic plasticity in hippocampal GABAergic neurons

Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin Repeat-rich Membrane Spanning) is a scaffold protein highly expressed in the nervous system. Previous work on neurons with altered Kidins220/ARMS expression suggested that this protein plays multiple roles in synaptic function. In this study, we analyzed the effects of Kidins220/ARMS ablation on basal synaptic transmission and on a variety of short-term plasticity paradigms in both excitatory and inhibitory synapses using a recently described Kidins220 full knockout mouse. Hippocampal neuronal cultures prepared from embryonic Kidins220(-/-) (KO) and wild type (WT) littermates were used for whole-cell patch-clamp recordings of spontaneous and evoked synaptic activity. Whereas glutamatergic AMPA receptor-mediated responses were not significantly affected in KO neurons, specific differences were detected in evoked GABAergic transmission. The recovery from synaptic depression of inhibitory post-synaptic currents in WT cells showed biphasic kinetics, both in response to paired-pulse and long-lasting train stimulation, while in KO cells the respective slow components were strongly reduced. We demonstrate that the slow recovery from synaptic depression in WT cells is caused by a transient reduction of the vesicle release probability, which is absent in KO neurons. These results suggest that Kidins220/ARMS is not essential for basal synaptic transmission and various forms of short-term plasticity, but instead plays a novel role in the mechanisms regulating the recovery of synaptic strength in GABAergic synapses.     

Regulated delivery of AMPA receptor subunits to the presynaptic membrane

In recent years, a role for AMPA receptors as modulators of presynaptic functions has emerged. We have investigated the presence of AMPA receptor subunits and the possible dynamic control of their surface exposure at the presynaptic membrane. We demonstrate that the AMPA receptor subunits GluR1 and GluR2 are expressed and organized in functional receptors in axonal growth cones of hippocampal neurons. AMPA receptors are actively internalized upon activation and recruited to the surface upon depolarization. Pretreatment of cultures with botulinum toxin E or tetanus toxin prevents the receptor insertion into the plasma membrane, whereas treatment with alpha-latrotoxin enhances the surface exposure of GluR2, both in growth cones of cultured neurons and in brain synaptosomes. Purification of small synaptic vesicles through controlled-pore glass chromatography, revealed that both GluR2 and GluR1, but not the GluR2 interacting protein GRIP, copurify with synaptic vesicles. These data indicate that, at steady state, a major pool of AMPA receptor subunits reside in synaptic vesicle membranes and can be recruited to the presynaptic membrane as functional receptors in response to depolarization.     

Synapsin is a novel Rab3 effector protein on small synaptic vesicles. II. Functional effects of the Rab3A-synapsin I interaction

Synapsins, a family of neuron-specific phosphoproteins that play an important role in the regulation of synaptic vesicle trafficking and neurotransmitter release, were recently demonstrated to interact with the synaptic vesicle-associated small G protein Rab3A within nerve terminals (Giovedi, S., Vaccaro, P., Valtorta, F., Darchen, F., Greengard, P., Cesareni, G., and Benfenati, F. (2004) J. Biol. Chem. 279, 43760-43768). We have analyzed the functional consequences of this interaction on the biological activities of both proteins and on their subcellular distribution within nerve terminals. The presence of synapsin I stimulated GTP binding and GTPase activity of both purified and endogenous synaptic vesicle-associated Rab3A. Conversely, Rab3A inhibited synapsin I binding to F-actin, as well as synapsin-induced actin bundling and vesicle clustering. Moreover, the amount of Rab3A associated with synaptic vesicles was decreased in synapsin knockout mice, and the presence of synapsin I prevented RabGDI-induced Rab3A dissociation from synaptic vesicles. The results indicate that an interaction between synapsin I and Rab3A exists on synaptic vesicles that modulates the functional properties of both proteins. Given the well recognized importance of both synapsins and Rab3A in synaptic vesicles exocytosis, this interaction is likely to play a major role in the modulation of neurotransmitter release.     

Using the atomic force microscope to study the interaction between two solid supported lipid bilayers and the influence of synapsin I

To measure the interaction between two lipid bilayers with an atomic force microscope one solid supported bilayer was formed on a planar surface by spontaneous vesicle fusion. To spontaneously adsorb lipid bilayers also on the atomic force microscope tip, the tips were first coated with gold and a monolayer of mercapto undecanol. Calculations indicate that long-chain hydroxyl terminated alkyl thiols tend to enhance spontaneous vesicle fusion because of an increased van der Waals attraction as compared to short-chain thiols. Interactions measured between dioleoylphosphatidylcholine, dioleoylphosphatidylserine, and dioleoyloxypropyl trimethylammonium chloride showed the electrostatic double-layer force plus a shorter-range repulsion which decayed exponentially with a decay length of 0.7 nm for dioleoylphosphatidylcholine, 1.2 nm for dioleoylphosphatidylserine, and 0.8 nm for dioleoyloxypropyl trimethylammonium chloride. The salt concentration drastically changed the interaction between dioleoyloxypropyl trimethylammonium chloride bilayers. As an example for the influence of proteins on bilayer-bilayer interaction, the influence of the synaptic vesicle-associated, phospholipid binding protein synapsin I was studied. Synapsin I increased membrane stability so that the bilayers could not be penetrated with the tip.     

Time-resolved fluorescence study of the neuron-specific phosphoprotein synapsin I. Evidence for phosphorylation-dependent conformational changes

Synapsin I is a major nerve terminal-specific phosphoprotein. It consists of a hydrophobic head region containing one phosphorylation site for either cAMP-dependent protein kinase or Ca2+/calmodulin-dependent protein kinase I and of a basic and elongated tail region containing two phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II. The steady-state emission spectrum of synapsin I was centered at 330 nm and was markedly red shifted upon denaturation, as expected for tryptophan residues segregated from the external aqueous environment in native conditions. Quenching studies showed a low accessibility of synapsin I tryptophans at low ionic strength which was further decreased by exposure to 200 mM NaCl but not significantly affected by phosphorylation. The intrinsic fluorescence of synapsin I was resolved into three major decay components with lifetimes of about 0.2, 3, and 7 ns. Upon phosphorylation of synapsin I on the tail sites, the spectra associated with the intermediate and long lifetimes were shifted to the red region, while the spectrum associated with the short lifetime was shifted to the blue region, in the absence of significant changes of the lifetimes. Phosphorylation of synapsin I on the head site was less effective. The anisotropy decay of synapsin I labeled with the long-living chromophore pyrene on Cys-223 was also analyzed. A shorter rotational correlation time was found for the tail phosphorylated form (corresponding to a Stokes radius of 41-42 A) than for the dephosphorylated or for the head phosphorylated form (corresponding to a Stokes radius of 60-63 A). The data suggest that phosphorylation of the tail sites induces changes in the conformation and hydrodynamic properties of synapsin I which may play a role in the regulation of the molecular interactions of synapsin I within the nerve terminal.     

Aspects of neural plasticity in the central nervous system—II. Numerical classification in neuroanatomy

The possibility of using taxonomic techniques to classify neuronal populations was explored. In particular, coefficients of similarity such as the Canberra metric and the Shannon diversity index were examined. The theoretical work in the field of numerical classification was adapted to the aim of characterizing various brain areas in classes according to their transmitter contents. The study of neuropeptide distribution in 15 brain areas clearly demonstrated that, of these, the hypothalamus is particularly noteworthy due to its higher neuropeptide content.