Dataset Information

Site-Specific Phosphorylation of VEGFR2 Is Mediated by Receptor Trafficking: Insights from a Computational Model.

ABSTRACT: Matrix-binding isoforms and non-matrix-binding isoforms of vascular endothelial growth factor (VEGF) are both capable of stimulating vascular remodeling, but the resulting blood vessel networks are structurally and functionally different. Here, we develop and validate a computational model of the binding of soluble and immobilized ligands to VEGF receptor 2 (VEGFR2), the endosomal trafficking of VEGFR2, and site-specific VEGFR2 tyrosine phosphorylation to study differences in induced signaling between these VEGF isoforms. In capturing essential features of VEGFR2 signaling and trafficking, our model suggests that VEGFR2 trafficking parameters are largely consistent across multiple endothelial cell lines. Simulations demonstrate distinct localization of VEGFR2 phosphorylated on Y1175 and Y1214. This is the first model to clearly show that differences in site-specific VEGFR2 activation when stimulated with immobilized VEGF compared to soluble VEGF can be accounted for by altered trafficking of VEGFR2 without an intrinsic difference in receptor activation. The model predicts that Neuropilin-1 can induce differences in the surface-to-internal distribution of VEGFR2. Simulations also show that ligated VEGFR2 and phosphorylated VEGFR2 levels diverge over time following stimulation. Using this model, we identify multiple key levers that alter how VEGF binding to VEGFR2 results in different coordinated patterns of multiple downstream signaling pathways. Specifically, simulations predict that VEGF immobilization, interactions with Neuropilin-1, perturbations of VEGFR2 trafficking, and changes in expression or activity of phosphatases acting on VEGFR2 all affect the magnitude, duration, and relative strength of VEGFR2 phosphorylation on tyrosines 1175 and 1214, and they do so predictably within our single consistent model framework.

SUBMITTER: Clegg LW

PROVIDER: S-EPMC4466579 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Site-Specific Phosphorylation of VEGFR2 Is Mediated by Receptor Trafficking: Insights from a Computational Model.

Clegg Lindsay Wendel LW Mac Gabhann Feilim F

PLoS computational biology 20150612 6

Matrix-binding isoforms and non-matrix-binding isoforms of vascular endothelial growth factor (VEGF) are both capable of stimulating vascular remodeling, but the resulting blood vessel networks are structurally and functionally different. Here, we develop and validate a computational model of the binding of soluble and immobilized ligands to VEGF receptor 2 (VEGFR2), the endosomal trafficking of VEGFR2, and site-specific VEGFR2 tyrosine phosphorylation to study differences in induced signaling b ...[more]

PMID: 26067165

Similar Datasets

Project description:N-methyl-d-aspartate receptors (NMDARs) are important for synaptic plasticity associated with many physiological functions and neurologic disorders. Protein kinase C (PKC) activation increases the phosphorylation and activity of NMDARs, and α2δ-1 is a critical NMDAR-interacting protein and controls synaptic trafficking of NMDARs. In this study, we determined the relative roles of PKC and α2δ-1 in the control of NMDAR activity. We found that α2δ-1 coexpression significantly increased NMDAR activity in HEK293 cells transfected with GluN1/GluN2A or GluN1/GluN2B. PKC activation with phorbol 12-myristate 13-acetate (PMA) increased receptor activity only in cells coexpressing GluN1/GluN2A and α2δ-1. Remarkably, PKC inhibition with Gӧ6983 abolished α2δ-1-coexpression-induced potentiation of NMDAR activity in cells transfected with GluN1/GluN2A or GluN1/GluN2B. Treatment with PMA increased the α2δ-1-GluN1 interaction and promoted α2δ-1 and GluN1 cell surface trafficking. PMA also significantly increased NMDAR activity of spinal dorsal horn neurons and the amount of α2δ-1-bound GluN1 protein complexes in spinal cord synaptosomes in wild-type mice, but not in α2δ-1 knockout mice. Furthermore, inhibiting α2δ-1 with pregabalin or disrupting the α2δ-1-NMDAR interaction with the α2δ-1 C-terminus peptide abolished the potentiating effect of PMA on NMDAR activity. Additionally, using quantitative phosphoproteomics and mutagenesis analyses, we identified S929 on GluN2A and S1413 (S1415 in humans) on GluN2B as the phosphorylation sites responsible for NMDAR potentiation by PKC and α2δ-1. Together, our findings demonstrate the interdependence of α2δ-1 and PKC phosphorylation in regulating NMDAR trafficking and activity. The phosphorylation-dependent, dynamic α2δ-1-NMDAR interaction constitutes an important molecular mechanism of synaptic plasticity.SIGNIFICANCE STATEMENT A major challenge in studies of protein phosphorylation is to define the functional significance of each phosphorylation event and determine how various signaling pathways are coordinated in response to neuronal activity to shape synaptic plasticity. PKC phosphorylates transporters, ion channels, and G-protein-coupled receptors in signal transduction. In this study, we showed that α2δ-1 is indispensable for PKC-activation-induced surface and synaptic trafficking of NMDARs, whereas the α2δ-1-NMDAR interaction is controlled by PKC-induced phosphorylation. Our findings reveal that α2δ-1 mainly functions as a phospho-binding protein in the control of NMDAR trafficking and activity. This information provides new mechanistic insight into the reciprocal roles of PKC-mediated phosphorylation and α2δ-1 in regulating NMDARs and in the therapeutic actions of gabapentinoids.

Project description:In all organisms, chromosome replication is regulated mainly at the initiation step. Most of the knowledge about the mechanisms that regulate replication initiation in bacteria has come from studies on rod-shaped bacteria, such as Escherichia coli and Bacillus subtilis Streptomyces is a bacterial genus that is characterized by distinctive features and a complex life cycle that shares some properties with the developmental cycle of filamentous fungi. The unusual lifestyle of streptomycetes suggests that these bacteria use various mechanisms to control key cellular processes. Here, we provide the first insights into the phosphorylation of the bacterial replication initiator protein, DnaA, from Streptomyces coelicolor We suggest that phosphorylation of DnaA triggers a conformational change that increases its ATPase activity and decreases its affinity for the replication origin, thereby blocking the formation of a functional orisome. We suggest that the phosphorylation of DnaA is catalyzed by Ser/Thr kinase AfsK, which was shown to regulate the polar growth of S. coelicolor Together, our results reveal that phosphorylation of the DnaA initiator protein functions as a negative regulatory mechanism to control the initiation of chromosome replication in a manner that presumably depends on the cellular localization of the protein.IMPORTANCE This work provides insights into the phosphorylation of the DnaA initiator protein in Streptomyces coelicolor and suggests a novel bacterial regulatory mechanism for initiation of chromosome replication. Although phosphorylation of DnaA has been reported earlier, its biological role was unknown. This work shows that upon phosphorylation, the cooperative binding of the replication origin by DnaA may be disturbed. We found that AfsK kinase is responsible for phosphorylation of DnaA. Upon upregulation of AfsK, chromosome replication occurred further from the hyphal tip. Orthologs of AfsK are exclusively found in mycelial actinomycetes that are related to Streptomyces and exhibit a complex life cycle. We propose that the AfsK-mediated regulatory pathway serves as a nonessential, energy-saving mechanism in S. coelicolor.

Project description:Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 in the N-terminal transactivation domain. In this study, the role of this phosphorylation site was investigated by characterizing the phosphorylation site mutant in the context of full length and truncated AR lacking the ligand-binding domain. The Y267F mutant showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. Expression of AR responsive genes in cells expressing truncated AR wt and AR-Y267F mutant under androgen deprived conditions was assessed by microarray gene expression analysis. The AR pathway score was increased in cells expressing truncated AR wt and decreased in cells expressing truncated AR-Y267F. These results support the concept that phosphorylation of Tyr-267 is required for AR nuclear translocation and recruitment and DNA binding and transcription of AR responsive genes. Gene expression profiling was performed using RNA samples from LNCaP cells expressing truncated AR-wt and truncated AR-Y267F growing in androgen deprived conditions. The RNA sample from vector control cells were used as reference sample. Two biological replicates were used per each cell line.

Dataset Information

Site-Specific Phosphorylation of VEGFR2 Is Mediated by Receptor Trafficking: Insights from a Computational Model.

Publications

Site-Specific Phosphorylation of VEGFR2 Is Mediated by Receptor Trafficking: Insights from a Computational Model.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets