7 results found
The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cuesand swiftly determines appropriate cellular responses. However, our understanding of cellulardecision-making has been limited by the small number of simultaneously monitored phospho-regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 condi-tions from a compilation of nearly 3 million phosphopeptide quantifications. This atlas identifiescommonly regulated kinases as those that are central in the signaling network and defines the logicrelationships between kinase pairs. Co-regulation along the conditions predicts kinase-complexand kinase-substrate associations. Additionally, the kinase regulation profile acts as a molecularfingerprint to identify related and opposing signaling states. Using this atlas, we identified essen-tial mediators of stem cell differentiation, modulators of Salmonella infection and new targets ofAKT1. This provides a global view of human phosphorylation-based signaling and the necessarycontext to better understand kinase driven decision-making.
Araujo AR, Gelens L, Sheriff RSM, et al., 2016, Positive Feedback Keeps Duration of Mitosis Temporally Insulated from Upstream Cell-Cycle Events, Molecular Cell, Vol: 64, Pages: 362-375, ISSN: 1097-4164
Cell division is characterized by a sequence of events by which a cell gives rise to two daughter cells. Quantitative measurements of cell-cycle dynamics in single cells showed that despite variability in G1-, S-, and G2 phases, duration of mitosis is short and remarkably constant. Surprisingly, there is no correlation between cell-cycle length and mitotic duration, suggesting that mitosis is temporally insulated from variability in earlier cell-cycle phases. By combining live cell imaging and computational modeling, we showed that positive feedback is the molecular mechanism underlying the temporal insulation of mitosis. Perturbing positive feedback gave rise to a sluggish, variable entry and progression through mitosis and uncoupled duration of mitosis from variability in cell cycle length. We show that positive feedback is important to keep mitosis short, constant, and temporally insulated and anticipate it might be a commonly used regulatory strategy to create modularity in other biological systems.
Johnson JR, Santos SD, Johnson T, et al., 2015, Prediction of functionally important phospho-regulatory events in xenopus laevis oocytes, PLOS Computational Biology, Vol: 11, ISSN: 1553-734X
The African clawed frog Xenopus laevis is an important model organism for studies in developmentaland cell biology, including cell-signaling. However, our knowledge of X. laevis proteinpost-translational modifications remains scarce. Here, we used a mass spectrometrybasedapproach to survey the phosphoproteome of this species, compiling a list of 2636phosphosites. We used structural information and phosphoproteomic data for 13 other speciesin order to predict functionally important phospho-regulatory events. We found that thedegree of conservation of phosphosites across species is predictive of sites with knownmolecular function. In addition, we predicted kinase-protein interactions for a set of cellcyclekinases across all species. The degree of conservation of kinase-protein interactionswas found to be predictive of functionally relevant regulatory interactions. Finally, usingcomparative protein structure models, we find that phosphosites within structured domainstend to be located at positions with high conformational flexibility. Our analysis suggeststhat a small class of phosphosites occurs in positions that have the potential to regulate proteinconformation.
Cruz C, Santos SD, Cabrita EJ, et al., 2013, Binding analysis between L-histidine immobilized and oligonucleotides by SPR and NMR., Int J Biol Macromol, Vol: 56, Pages: 175-180
Saturation transfer difference (STD) NMR technique and surface plasmon resonance (SPR) are used to study amino acid affinity supports-nucleotides interactions with L-histidine amino acid immobilized on a surface as model support. We have immobilized L-histidine ligand on a carboxymethyldextran-modified gold surface intended for surface plasmon resonance and we analyze the binding profiles of synthetic polynucleotides (1-6 base, sugar and backbone) by determining the equilibrium dissociation constant (KD). The SPR binding profile (square-shaped) is identical for all the complexes and the highest binding affinity can be found for polyA₆ followed by polyG₆. As expected, the 5'-mononucleotides have the lowest affinity. To further study the structural aspects of the interaction we investigate the polynucleotide binding preferences to L-histidine chromatography support by STD-NMR spectroscopy. These results revealed that an increase in the number of bases and backbone to 6 units leads to more contacts with the support, where the main driving force for the interaction with polynucleotides are through the base, except for polyC₆, which is mainly through sugar-phosphate backbone. Therefore, the combination of SPR measurements with STD-NMR technique allowed to establish fine details of the molecular recognition process involved in amino acid affinity supports-nucleotides complexes.
Catarino T, Ribeiro L, Santos SD, et al., 2013, Regulation of synapse composition by protein acetylation: the role of acetylated cortactin., J Cell Sci, Vol: 126, Pages: 149-162
Protein acetylation affects synaptic plasticity and memory, but its effects on synapse composition have not been addressed. We found that protein acetylation promotes the dendritic clustering of the excitatory postsynaptic scaffold protein PSD95 in hippocampal neurons, without affecting the total levels of this protein. Cortactin, an F-actin-binding protein enriched in dendritic spines, is a substrate for acetylation and has a role in spine morphogenesis. Recent studies showed that cortactin acetylation changes its ability to bind F-actin and regulates cellular motility, but the function of cortactin acetylation in neuronal cells is so far unknown. We tested whether acetylation of cortactin influences its morphogenic function by overexpressing wild-type cortactin, or the mimetic mutants for acetylated or deacetylated cortactin, in hippocampal neurons, and found that cortactin acetylation has an impact on PSD95 clustering, independent from its function as actin dynamics regulator. Moreover, acetylated cortactin can rescue the reduction in PSD95 clustering mediated by knockdown of cortactin. We also found that acetylation of cortactin is correlated with decreased cortactin interaction with p140Cap and Shank1, and with lower cortactin phosphorylation at tyrosine 421. The neurotrophin BDNF promoted the acetylation of cortactin in hippocampal neurons, suggesting that BDNF may regulate excitatory synapses and PSD95 dendritic clustering at least in part by changing the acetylation level of cortactin. Our findings unravel an unsuspected role for cortactin acetylation in the regulation of PSD95 dendritic clustering, which may work in concert with cortactin's role in spine development.
Santos SDM, Wollman R, Meyer T, et al., 2012, Spatial positive feedback at the onset of mitosis., Cell, Vol: 149, Pages: 1500-1513
Mitosis is triggered by the activation of Cdk1-cyclin B1 and its translocation from the cytoplasm to the nucleus. Positive feedback loops regulate the activation of Cdk1-cyclin B1 and help make the process irreversible and all-or-none in character. Here we examine whether an analogous process, spatial positive feedback, regulates Cdk1-cyclin B1 redistribution. We used chemical biology approaches and live-cell microscopy to show that nuclear Cdk1-cyclin B1 promotes the translocation of Cdk1-cyclin B1 to the nucleus. Mechanistic studies suggest that cyclin B1 phosphorylation promotes nuclear translocation and, conversely, nuclear translocation promotes cyclin B1 phosphorylation, accounting for the feedback. Interfering with the abruptness of Cdk1-cyclin B1 translocation affects the timing and synchronicity of subsequent mitotic events, underscoring the functional importance of this feedback. We propose that spatial positive feedback ensures a rapid, complete, robust, and irreversible transition from interphase to mitosis and suggest that bistable spatiotemporal switches may be widespread in biological regulation.
Santos SD, Iuliano O, Ribeiro L, et al., 2012, Contactin-associated protein 1 (Caspr1) regulates the traffic and synaptic content of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors., J Biol Chem, Vol: 287, Pages: 6868-6877
Glutamate receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type mediate fast excitatory synaptic transmission in the CNS. Synaptic strength is modulated by AMPA receptor binding partners, which regulate receptor synaptic targeting and functional properties. We identify Contactin-associated protein 1 (Caspr1) as an AMPA receptor interactor. Caspr1 is present in synapses and interacts with AMPA receptors in brain synaptic fractions. Coexpression of Caspr1 with GluA1 increases the amplitude of glutamate-evoked currents. Caspr1 overexpression in hippocampal neurons increases the number and size of synaptic GluA1 clusters, whereas knockdown of Caspr1 decreases the intensity of synaptic GluA1 clusters. Hence, Caspr1 is a regulator of the trafficking of AMPA receptors to synapses.
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