Dataset Information

Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions.

ABSTRACT: The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases.

SUBMITTER: Courcelles M

PROVIDER: S-EPMC4188273 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions.

Courcelles Mathieu M Frémin Christophe C Voisin Laure L Lemieux Sébastien S Meloche Sylvain S Thibault Pierre P

Molecular systems biology 20130528

The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics an ...[more]

PMID: 23712012

Similar Datasets

Project description:Diabetic nephropathy is the most common chronic kidney disease in the world and the main cause of end-stage renal disease (ESRD). The structural integrity of podocytes is fundamental to the normal function of the glomerulus, and the role of glycogen synthase kinase 3β (GSK-3β) in podocytes is complicated. A thorough understanding of GSK-3β is crucial to understand the mechanism of diabetic nephropathy. To analyze the roles of GSK-3β in podocytes, GSK-3β knockdown lentivirus by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)9 was applied to establish stable cell lines. Mass spectrometry was utilized to search for differentially expressed proteins. Consequently, we found 34 proteins with higher levels and 115 proteins with lower levels in GSk-3β knockdown cells than in control cells and identified 581 phosphosites with higher phosphorylation levels and 288 phosphosites with lower phosphorylation levels. We performed functional enrichment analysis of these proteins and phosphorylated proteins based on public databases. Enrichment analysis revealed that GSK-3β participates in the spliceosome, Hippo signaling pathway, actin binding, structural molecule activity, and other pathways. Then, we used motif analysis of phosphate sites to determine 89 conserved motifs based on 1,068 phosphoserine (pS) sites and 15 conserved motifs in view of 104 phosphothreonine (pT) sites. Additionally, protein-protein interaction network analysis was carried out using the STRING database. Cytoscape's add-on Molecular Complex Detection (MCODE) was used to analyze key and core protein groups. In quantitative differential protein analysis, four MCODEs were obtained, and 22 MCODEs were obtained in the analysis of the phosphoproteome of differentially expressed proteins. Finally, we analyzed the kinase regulatory network in podocytes after GSK-3β knockdown and identified 299 protein kinases and 3,460 significantly changed phosphorylation modification sites on 1,574 proteins. These results will be valuable for further research on GSK-3β.

Project description:The norepinephrine transporter (NET) mediates the clearance of norepinephrine (NE) from the extracellular space and is a target of therapeutic antidepressants and psychostimulants. Previously we identified a MAP kinase phosphatase 3 (MKP3), as an important modulator of protein kinase C (PKC) mediated internalization of the related dopamine transporter (DAT). Here we show that MKP3 decreases PKC-mediated down regulation of NET expressed in PC12 cells. We demonstrate that this process involves a PKC-stimulated decrease of NET surface expression that is dependent on dynamin. Surprisingly, MAP kinase inhibitors have no effect on the PKC-mediated regulation of NET activity, suggesting that, like PKC-mediated regulation of the DAT, the acute activation of MAP kinases is not likely to be involved. To elucidate potential mechanisms we used a substrate trap-based assay to identify extracellular-signal-regulated kinase (ERK)1/2 as the predominant substrate of MKP3. Furthermore we also established that brief chemical stabilization of a modified destabilized MKP3 does not alter PKC-mediated down regulation of NET. Finally, the expression of a dominant negative version of H-Ras, an upstream activator of ERK1/2, abolishes phorbol 12-myristate 13-acetate (PMA)-mediated down regulation of NET in a manner similar to MKP3. Taken together we propose that chronic MKP3 expression regulates surface NET through the sustained inhibition of ERK1/2 MAP kinase signaling that alters gene expression in PC12 cells. This is supported by gene expression data from naïve and MKP3-expressing PC12 cells that reveal robust decreases in gene expression of several genes in the MKP3-tranfected cells. Interestingly, caveolin-1, a protein with a critical role in membrane protein trafficking is down regulated by MKP3 expression. We further show that selective silencing of the caveolin-1 gene in naïve PC12 cells attenuates PKC-mediated downregulation of NET activity, consistent with a potential role for caveolins in regulating NET surface expression. In summary, these results suggest that chronic MKP3 expression alters the expression of genes in PC12 cells that are involved in the regulation of NET surface expression.

Dataset Information

Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions.

Publications

Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure