Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Filamin C domain 18-21 in vitro kinase assay

ABSTRACT: In vitro kinase assay with human filamin C domain 18-21 recombinantly expressed in E.coli, purified by Ni2+-NTA beads and incubated with PKC alpha, Akt or a combination of both to identify kinase specific phosphorylation sites.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

SUBMITTER: Friedel Drepper

LAB HEAD: Bettina Warscheid

PROVIDER: PXD008893 | Pride | 2020-05-25

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Publications

Phosphoproteomics identifies dual-site phosphorylation in an extended basophilic motif regulating FILIP1-mediated degradation of filamin-C.

Reimann Lena L Schwäble Anja N AN Fricke Anna L AL Mühlhäuser Wignand W D WWD Leber Yvonne Y Lohanadan Keerthika K Puchinger Martin G MG Schäuble Sascha S Faessler Erik E Wiese Heike H Reichenbach Christa C Knapp Bettina B Peikert Christian D CD Drepper Friedel F Hahn Udo U Kreutz Clemens C van der Ven Peter F M PFM Radziwill Gerald G Djinović-Carugo Kristina K Fürst Dieter O DO Warscheid Bettina B

Communications biology 20200522 1

The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig- ...[more]

PMID: 32444788

Similar Datasets

FLNc S2233 S2236 PRM analysis

Project description:Skeletal muscle is known to adapt dynamically to changes in workload by regulatory processes of the phosphatidylinositide 3-kinase (PI3K)/Akt pathway. We performed a global quantitative phosphoproteomics analysis of contracting mouse C2 myotubes treated with insulin growth factor 1 (IGF-1) or LY294002 to activate or inhibit PI3K/Akt signaling, respectively. Among the significantly regulated phosphopeptides we identified the novel extended basophilic motif RxRxxp[S/T]xxp[S] to be enriched in the set of down-regulated phosphopeptides following inhibition of PI3K/Akt signaling. Using literature-based text mining we identified the kinases Akt, serum and glucocorticoid-regulated kinase 1 (SGK1) and p70S6 kinase to be potentially involved in the phosphorylation of the first serine in the RxRxxp[S/T]xxp[S] motif, whereas no kinase targeting the serine in the +3 position was revealed. In the signaling adapter protein filamin c (FLNc) we found this novel motif in immunoglobulin (Ig)-like domain 20 which is involved in various protein interactions. Through in vitro and in cellulo kinase assays we identified Akt and protein kinase C alpha as the responsible kinases phosphorylating FLNc in this motif at the first and the second serine, respectively.

2020-06-30 | PXD009228 | Pride

Phosphoproteomics Identifies Dual-Site Phosphorylation in an Extended Basophilic Motif Regulating FILIP1-mediated Degradation of filamin-C FLNc d18-21 BioID experiment

Project description:FLNc, the muscle-specific isoform of the filamin family, is a multi-adaptor protein, comprising 1 amino-terminal actin-binding (ABD) domain followed by 24 immunoglobulin-like (Ig) domains. While FLNc can form homodimers via the last Ig-like domain and thus function as an actin-crosslinker like the other filamins, it features a unique insertion of 82 amino acids (aa) in domain 20. This insert was not only shown to mediate the interaction to several FLNc binding partners, but also to contain an Akt-mediated phosphorylation site at S2234 of mouse FLNc (mFLNc). To reveal novel proteins in the nano-environment of FLNc within myotubes under mild electrical pulse stimulation conditions, we applied a quantitative BioID appraoch.

2021-07-12 | PXD009159 | Pride

FilaminC pull-down experiment from myotube lysates

Project description:This project was designed to identify a difference in the binding affinity of FILIP1 to different FLNc variants. To this end FLNc domain 1-3 and FLNc d18-21 were used as bait for a pull-down analysis.

2020-05-25 | PXD017670 | Pride

Large-scale in vitro kinase assay with FLNc d23-24 and PKC alpha

Project description:Large-scale in vitro kinase assay with human and murine FLNc and its site mutants and PKC alpha.

2017-01-02 | PXD003185 | Pride

H3K79 methyltransferase Dot1p/DOT1L is phosphorylated by proline-directed MAP kinases Hog1p and p38/MAPK11

Project description:In vitro phosphorylation assays were conducted to identify phosphorylation sites on Dot1p catalysed by Hog1p. As several other kinases were co-purified with Hog1p, dead kinase control assays (Hog1p-K52R and Hog1p-D144A) were performed to verify the identified Dot1p phospho-sites. This entry contains the data pertaining to the in vitro Hog1p dead kinase phosphorylation assays, which is in supplement to existing PRIDE entry PXD036747.

2024-01-29 | PXD040407 | Pride

H3K79 methyltransferase Dot1p/DOT1L is phosphorylated by proline-directed MAP kinases Hog1p and p38/MAPK11

Project description:Disruptor of telomeric silencing (Dot1p) is an exquisitely conserved histone methyltransferase, and is the sole enzyme responsible for H3K79 methylation in the budding yeast, Saccharomyces cerevisiae. It has been shown to be highly phosphorylated in vivo; however, the upstream kinases that act on Dot1p are unknown in yeast and all other eukaryotes. Here, we used in vitro and in vivo kinase discovery approaches to show that the mitogen-activated protein kinase Hog1p is a bona fide kinase of Dot1p methyltransferase. In in vitro kinase assays, Hog1p phosphorylates Dot1p at multiple sites, including at several proline-adjacent sites that are consistent with its known substrate preference. Hog1p kinase activity was specifically enhanced at these proline-adjacent sites on Dot1p upon its activation by the osmostress-responsive kinase, Pbs2p. We then show that genomic deletion of HOG1 markedly reduces phosphorylation at specific sites on Dot1p in vivo, thus providing evidence for Hog1p kinase activity on Dot1p in budding yeast cells. Phenotypic analysis of knockout and phosphosite mutant yeast strains revealed the importance of Hog1p-catalysed phosphorylation of Dot1p for cellular responses to ultraviolet-induced DNA damage. This kinase-substrate relationship is conserved in mammalian systems, where human DOT1L (ortholog of Dot1p) can be phosphorylated by the proline-directed kinase p38β/MAPK11 (ortholog of Hog1p) at multiple sites in vitro. Taken together, our findings establish Hog1p and p38β/MAPK11 as the first kinases known to act on H3K79 methyltransferase enzymes in any eukaryotic species.

2024-01-29 | PXD036747 | Pride

Identification of soybean seed developmental stage specific and tissue specific miRNA targets by degradome sequencing .

Project description:Five degradome libraries were constructed from three different seed developmental stages. Separate degradome libraries were constructed for seed coat and cotyledons to identify the tissue specific miRNAs and their potential targets. Sequencing and analysis of degradome libraries gives identification of 183 different targets for 80 known soybean miRNAs. We found 30 cotyledon specific, 18 seed coat specific and 32 miRNAs found in both tissues irrespective of the developmental stages. One interesting observation is that we found more miRNA targets in late seed developmental stages than earlier stages. Additionally, we have validated four different auxin response factor genes as targets for gma-miR160 via RNA ligase mediated 5? rapid amplification of cDNA ends (RLM-5?RACE). GO analysis indicated the enrichment of miRNA target genes in seed development. Construction of degradome libraries using cotyledons and seed coats from 3 different developmental stages

2012-08-07 | E-GEOD-34433 | biostudies-arrayexpress

Phosphorylation of multiple proteins involved in ciliogenesis by Tau Tubulin kinase 2

Project description:Abstract Primary cilia are organelles necessary for proper implementation of developmental and homeostasis processes. To initiate their assembly, coordinated actions of multiple proteins are needed. Tau tubulin kinase 2 (TTBK2) is a key player in the cilium assembly pathway, controlling final step of cilia initiation. The function of TTBK2 in ciliogenesisis critically dependent on its kinase activity, however, precise mechanism of action of this kinase is so far incompletely understood, in part due to very limited information about its relevant substrates. In this study we identify CEP83, CEP89, CCDC92, Rabin8 and DVL3 as substrates of TTBK2 kinase activity. Further, we characterise a set of phosphosites of the newly identified substrates and CEP164, induced by TTBK2 in vitro and in vivo and show that TTBK2 preferentially phosphorylates examined substrates at intrinsically disordered regions (IDRs). Intriguingly, we further show that identified TTBK2 phosphosites and consensus sequence delineated from those are distinct from motifs previously assigned to TTBK2. Finally, we address functional relevance of selected phosphorylations of CEP164 and provide evidence that the examined TTBK2-induced phosphorylations of CEP164 are relevant for the process of cilia formation. In summary, our work provides important insight into substrates-TTBK2 kinase relationship and suggests that multiple phosphorylation of IDRs is probably involved in the control of ciliogenesis in human cells.

2020-03-25 | PXD014199 | Pride

Comparative Phosphoproteomic Profiling of Type III Adenylyl Cyclase Knockout and Control, Male and Female Mice Prefrontal Cortex

Project description:Type III adenylyl cyclase (AC3, ADCY3) is predominantly enriched in neuronal primary cilia throughout the central nervous system (CNS). Genome-wide association studies in humans have associated ADCY3 with major depressive disorder and autistic spectrum disorder, both of which exhibit sexual dimorphism. To date, it is unclear how AC3 affects protein phosphorylation and signal networks in central neurons, and what causes the sexual dimorphism of autism. We employed a mass spectrometry (MS)-based phosphoproteomic approach to quantitatively profile differences in phosphorylation between inducible AC3 knockout (KO) and wild type (WT), male and female mice. In total, we identified 4655 phosphopeptides from 1756 proteins, among which 565 phosphopeptides from 322 proteins were repetitively detected in all samples. Comparison of AC3 KO and WT datasets revealed that phosphopeptides with motifs matching proline-directed kinases’ recognition sites had a lower abundance in the KO dataset than in WTs. We detected 14 phosphopeptides restricted to WT dataset (i.e., from Spast and Ppp1r14a) and 35 exclusively in KOs (i.e., from Sptan1, Arhgap20, Arhgap44, and Pde1b). Moreover, 95 phosphopeptides were identified only in female dataset and 26 only in males. Label-free MS spectrum quantification using Skyline further identified phosphopeptides that had high expression abundance in each sample group. In total, over 200 modifications were gender-biased and had increased expression in females relative to males. 29% gender-biased phosphopeptides were from autism-associated proteins, including Dlg1, Dlg4, Dlgap2, Syn1, Syngap1, Ctnna1, Ctnnd1, Ctnnd2, Pkp4, and Arvcf. These autism proteins were well-connected in protein-protein interaction network that centered around Dlg4. This study provided the first phosphoproteomics evidence, suggesting that gender-biased post-translational phosphorylation may be implicated in the sexual dimorphism of autism.

2019-04-10 | PXD012259 | Pride

Chemical Genetic Identification of Cyclin-G-associated kinase (GAK) Substrates Reveals its Role in Regulating Na+/K+-ATPase and Sipa1L1 in Neurons

Project description:Recent genetic evidence implicates the serine/threonine kinase cyclin G-associated kinase (GAK) as a Parkinson’s disease risk. However, its role in neuronal function and many downstream effectors remain unclear. Employing a chemical genetics method, we show here that the sodium potassium pump (Na+/K+-ATPase) is a GAK target in the brain. We further show that GAK modulates Na+/K+-ATPase at a novel site affecting both pump localization and function. Whole-cell patch clamp recordings from CA1 pyramidal cells in GAK conditional knockout mice show a larger change in resting membrane potential when exposed to the Na+/K+-ATPase blocker, ouabain, indicating altered Na+/K+-ATPase function in GAK knockouts. Additionally, we show that GAK-deficient neurons have enlarged dendritic spines and we identify the spine associated protein Sipa1L1 (or SPAR) as a GAK target, which may contribute to this effect. Our results reveal novel functions of GAK in neurons.

2019-01-07 | PXD011319 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data