Systematic investigation of hierarchical phosphorylation by protein kinase CK2

St‐Denis, Nicole; Gabriel, Michelle; Turowec, Jacob P.; Gloor, Gregory B.; Li, Shawn S.‐C.; Gingras, Anne‐Claude; Litchfield, David W.

doi:10.1016/j.jprot.2014.10.020

Cited by 69 publications

(71 citation statements)

References 42 publications

(56 reference statements)

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“…Furthermore, we observed that the C-tail of FGF14 contains a consensus GSK–3 phosphorylation motif ( S/T )XXX(S/T), the first residue in this sequence corresponding to S226. (The phosphorylation motif for GSK–3 contains two serine/threonines: the first, at the P position, corresponds to the site phosphorylated by GSK–3, and the second, at the P + 4 position, corresponds to a priming phosphorylation site that increases the efficiency of GSK–3-dependent phosphorylation (Casaday et al, 2004; St-Denis et al, 2015; ter Haar et al, 2001)). Based on these observations, we conducted an in vitro phosphorylation assay using recombinant GSK–3β with a FGF14 peptide with the sequence KPGVTP S KSTSASAIMNGGK.…”

Section: Resultsmentioning

confidence: 99%

PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

Hsu

Wildburger

Haidacher

et al. 2017

Experimental Neurology

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Background Cognitive impairment in humans with Alzheimer's disease (AD) and in animal models of Aβ-pathology can be ameliorated by treatments with the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ) agonists, such as rosiglitazone (RSG). Previously, we demonstrated that in the Tg2576 animal model of AD, RSG treatment rescued cognitive deficits and reduced aberrant activity of granule neurons in the dentate gyrus (DG), an area critical for memory formation. Methods We used a combination of mass spectrometry, confocal imaging, electrophysiology and split-luciferase assay and in vitro phosphorylation and Ingenuity Pathway Analysis. Results Using an unbiased, quantitative nano-LC-MS/MS screening, we searched for potential molecular targets of the RSG-dependent rescue of DG granule neurons. We found that S226 phosphorylation of fibroblast growth factor 14 (FGF14), an accessory protein of the voltage-gated Na+ (Nav) channels required for neuronal firing, was reduced in Tg2576 mice upon treatment with RSG. Using confocal microscopy, we confirmed that the Tg2576 condition decreased PanNav channels at the AIS of the DG, and that RSG treatment of Tg2576 mice reversed the reduction in PanNav channels. Analysis from previously published data sets identified correlative changes in action potential kinetics in RSG-treated T2576 compared to untreated and wildtype controls. In vitro phosphorylation and mass spectrometry confirmed that the multifunctional kinase GSK–3β, a downstream target of insulin signaling highly implicated in AD, phosphorylated FGF14 at S226. Assembly of the FGF14:Nav1.6 channel complex and functional regulation of Nav1.6-mediated currents by FGF14 was impaired by a phosphosilent S226A mutation. Bioinformatics pathway analysis of mass spectrometry and biochemistry data revealed a highly interconnected network encompassing PPARγ, FGF14, SCN8A (Nav 1.6), and the kinases GSK–3 β, casein kinase 2β, and ERK1/2. Conclusions These results identify FGF14 as a potential PPARγ-sensitive target controlling Aβ-induced dysfunctions of neuronal activity in the DG underlying memory loss in early AD.

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Section: Resultsmentioning

confidence: 99%

PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

Hsu

Wildburger

Haidacher

et al. 2017

Experimental Neurology

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“…CK2 is known to phosphorylate its substrates in clusters, with phosphorylation at one site priming the substrate for phosphorylation at additional sites (27,28). The C-terminal region contains two potential Ser clusters: a proximal cluster of S503, S505, and S518 and a distal cluster of S572, S579, and S584 (Fig.…”

Section: Ecd Levels and Localization Do Not Change During Cell Cycle mentioning

confidence: 99%

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression

Mir

Bele

Mirza

et al. 2016

Molecular and Cellular Biology

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Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. P recisely regulated cell proliferation is essential for embryonic development as well as homeostasis in adult organs and tissues, whereas uncontrolled cell proliferation is a hallmark of cancer (1). A more in-depth understanding of the regulatory controls of cell cycle progression is therefore of great interest.The Ecd gene was originally inferred from studies of Drosophila melanogaster ecdysoneless (or ecd) mutants that exhibit defective development due to reduced production of the steroid hormone ecdysone (2). Subsequent cloning of drosophila ecd helped identify a cell-autonomous role of ECD protein in cell survival aside from its non-cell-autonomous role in ecdysis (molting) (3). However, the molecular basis of how ECD functions remains unknown (3). The human ECD homologue was initially identified in a screen of human open reading frames that complemented the S. cerevisiae mutants lacking Gcr2 (glycolysis regulation 2) gene, and it rescued the growth defect caused by reduced glycolytic enzyme activity in Gcr2 mutants. The human gene was initially designated HSGT1 (human suppressor of Gcr2) and was suggested to function as a coactivator of glycolytic gene transcription (4). However, ECD protein bears no structural homology to Gcr2, and a true ECD orthologue is absent in S. cerevisiae, suggesting that ECD likely functions by distinct mechanisms.We identified human ECD in a yeast two-hybrid screen of human mammary epithelial cell cDNA-encoded proteins for novel binding partners of the human papillomavirus 16 (HPV16) E6 oncogene (5). We showed that deletion of Ecd gene in mice causes embryonic lethality, identifying an esse...

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“…FMRP S499 could be regulated by dephosphorylation alone; however, it has been shown that S499 phosphorylation levels are not affected by PP2A, the putative FMRP phosphatase (Bartley et al, 2014). A recent exhaustive study has shown that although CK2 is constitutively active, it can promote secondary hierarchical phosphorylation by other kinases, many of which are regulated in a signal-dependent manner (St-Denis et al, 2015). This supported our hypothesis that phosphorylation of FMRP S499 is permissive for secondary phosphorylation of FMRP.…”

Section: Introductionmentioning

confidence: 99%

Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

Bartley

O’Keefe

Blice-Baum

et al. 2016

eNeuro

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Systematic investigation of hierarchical phosphorylation by protein kinase CK2

Cited by 69 publications

References 42 publications

PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression

Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

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