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CDKL5 substrates - Phosphoproteomic screening identifies physiological substrates of the CDKL5 kinase

ABSTRACT: Identification of CDKL5 substrates. Immuno-precipitated proteins were TMT labelled and analysed via LC-MS/MS.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Osteosarcoma

SUBMITTER: John Rouse

LAB HEAD: John Rouse

PROVIDER: PXD009327 | Pride | 2018-10-02

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	171108_Uniprot_homo_sapiens.fasta	Fasta
	180307_FW013-CEP131.xml	Xml
	180307_FW013-DLG5.xml	Xml
	180307_FW013-MAP1S-II.xml	Xml
	CEP131.rar	Other

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Publications

Phosphoproteomic screening identifies physiological substrates of the CDKL5 kinase.

Muñoz Ivan M IM Morgan Michael E ME Peltier Julien J Weiland Florian F Gregorczyk Mateusz M Brown Fiona Cm FC Macartney Thomas T Toth Rachel R Trost Matthias M Rouse John J

The EMBO journal 20180928 24

Mutations in the gene encoding the protein kinase CDKL5 cause a debilitating neurodevelopmental disease termed CDKL5 disorder. The impact of these mutations on CDKL5 function is poorly understood because the substrates and cellular processes controlled by CDKL5 are unclear. Here, we describe a quantitative phosphoproteomic screening which identified MAP1S, CEP131 and DLG5-regulators of microtubule and centrosome function-as cellular substrates of CDKL5. Antibodies against MAP1S phospho-Ser<sup>9 ...[more]

PMID: 30266825

Similar Datasets

Quantitative phosphoproteomic for CDKL5 substrates

Project description:Global phosphoproteomic screen to identify the first cellular substrates of CDKL5. CDKL5 knock-out U2OS cells and CDKL5 wt U2OS cells were generated for the TMT-based phosphoproteomic. Thi leads to the identification and further validation of several phosphopetides of MAP1S, CEP131 and CDKL5 itself. The phosphoproteomic analysis allowed the identification of the first cellular substrates for CDKL5 kinase.

2018-09-24 | PXD009374 | Pride

Phosphoproteomic screening identifies physiological substrates of the CDKL5 kinase.

Project description:Mutations in the gene encoding the protein kinase CDKL5 cause a debilitating neurodevelopmental disease termed CDKL5 disorder. The impact of these mutations on CDKL5 function is poorly understood because the substrates and cellular processes controlled by CDKL5 are unclear. Here, we describe a quantitative phosphoproteomic screening which identified MAP1S, CEP131 and DLG5-regulators of microtubule and centrosome function-as cellular substrates of CDKL5. Antibodies against MAP1S phospho-Ser900 and CEP131 phospho-Ser35 confirmed CDKL5-dependent phosphorylation of these targets in human cells. The phospho-acceptor serine residues in MAP1S, CEP131 and DLG5 lie in the motif RPXSA, although CDKL5 can tolerate residues other than Ala immediately C-terminal to the phospho-acceptor serine. We provide insight into the control of CDKL5 activity and show that pathogenic mutations in CDKL5 cause a major reduction in CDKL5 activity in vitro and in cells. These data reveal the first cellular substrates of CDKL5, which may represent important biomarkers in the diagnosis and treatment of CDKL5 disorder, and illuminate the functions of this poorly characterized kinase.

| S-EPMC6293279 | biostudies-literature

Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics

Project description:Loss-of-function mutations in CDKL5 kinase causes severe neurodevelopmental delay and early-onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule-associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3-labelled plus-end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAP1S dissociation from microtubules. Additionally, anterograde cargo trafficking is compromised in CDKL5 knockout mouse dendrites. Finally, EB2 phosphorylation is reduced in patient-derived human neurons. Our results reveal a novel activity-dependent molecular pathway in dendritic microtubule regulation and suggest a pathological mechanism which may contribute to CDKL5 deficiency disorder.

2018-09-18 | PXD010511 | Pride

Expression profiling in WT vs CDKL5 mutated IPSCs

Project description:Mutations in the human CDKL5 gene have been associated with early onset seizure variant of Rett Syndrome. In order to investigate the potential involvement of CDKL5 in the regulation of gene expression, we compared expression profiles in WT vs CDKL5-mutated IPS clones from two patients (one male and one female) with different mutations using two-colour microarray experiments. For the female patient (CDKL5 mutation p.Gln347X) we compared one clone expressing the mutant CDKL5 allele to another clone from the same patient that expresses the wild-type allele. Maintenance of X chromosome inactivation and the resulting mono-allelic expression of CDKL5 were confirmed by androgen receptor assay and direct sequencing of CDKL5 mRNA. The clone derived from the male patient (CDKL5 mutation p.Thr288Ile) was compared to a clone derived from a normal newborn male. For each mutant/control pair four technical replicates were performed for a total of eight chip hybridizations.

2014-07-02 | E-MTAB-2223 | biostudies-arrayexpress

Nuclear substrates of the CDKL5 kinase - Phosphoproteomics

Project description:Identification of nuclear substrates of the CDKL5 kinase by quantitative phosphoproteomics

2020-12-10 | PXD022916 | Pride

Transcription profiling by array of human clonal primary fibroblasts from patients with CDKL5 mutations

Project description:Mutations in the human CDKL5 gene have been shown to cause infantile spasms, as well as Rett syndrome-like phenotype. Because CDKL5 is subjected to X chromosome inactivation (XCI), individual cells from CDKL5 mutation girls either express the wild-type or mutant allele, likely resulting in different consequences at both the cellular and molecular levels. To identify these consequences, we carried out gene expression profiling on clonal populations derived from primary cultures of three patientsM-^R fibroblasts expressing either allele. A total of 16 up-regulated and 20 down-regulated genes were identified. The differentially expressed gene products, mostly involved in differentiation and oxidative stress may be related to a mechanism underlying mental retardation and epilepsy. Among these differentially expressed proteins, the apoptosis signal-regulated kinase MAP3K5 expression was found to be altered in non-neuronal, but also in neuronal CDKL5 deficient cells. Due to the fact that MAP3K5 activates MAP kinase pathway, which mediates signals leading to both differentiation and survival in neuronal cells, we suggest that a CDKL5 deficit may induce changes in synaptic plasticity in patientM-^Rs brain.

2011-07-23 | E-MEXP-2833 | biostudies-arrayexpress

Artificial escape from XCI by DNA methylation editing of the CDKL5 gene [RNA-seq]

Project description:A significant number of X-linked genes escape from X-chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we sought to create an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels above 60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X-chromosome inactivation. Understanding the epigenetics associated with escape from X-chromosome inactivation has potential for those suffering from X-linked disorders.

2020-01-01 | GSE137663 | GEO

Artificial escape from XCI by DNA methylation editing of the CDKL5 gene [methylation array]

2020-01-01 | GSE137541 | GEO

Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa (Nimblegen)

Project description:In higher eukaryotes, histone methylation is involved in the maintenance of cellular identity during somatic development. During spermatogenesis, most nucleosomes are replaced by protamines. Therefore, it is unclear if histone modifications function in paternal transmission of epigenetic information. Here we show that active H3K4 di-methylation (H3K4me2) and repressive H3K27 tri-methylation (H3K27me3), two modifications important for Trithorax and Polycomb-mediated gene regulation, are present in chromatin of human spermatozoa and show methylation-specific distributions at regulatory regions. H3K4me2-marked promoters control gene functions in spermatogenesis and cellular homeostasis suggesting that this mark reflects germline transcription. In contrast, H3K27me3 marks promoters of key developmental regulators in sperm as in soma. Many H3K27me3-marked genes are never expressed in the male and female germline, and in early “totipotent” embryos, suggesting a function for Polycomb in repressing somatic determinants across generations. Targets of H3K4me2 and H3K27me3 are also modified in mouse spermatozoa, implicating an evolutionary conserved role for histone methylation in chromatin inheritance via the male germline. Chromatin immuno precipitation (ChIP) was performed on sperm samples obtained from 9 normospermic donors. DNA associated with H3K4me2 or H3K27me3 was precipitated using specific antibodies. Input and precipitated DNA were amplified and hybridized to a tiling microarray (NimbleGen Systems Inc.) representing 18029 promoter regions (2200bp upstream to 500bp downstream of transcription start sites) of all RefSeq annotated human genes. For each modification 3 independent ChIP experiments were performed of which one was hybridized in a dye swap configuration.

2010-05-16 | E-GEOD-13863 | biostudies-arrayexpress

RpoHI and RpoHII regulons in Rhodobacter sphaeroides 2.4.1 from chromatin immuno-precipitation

Project description:In this study, we performed a ChIP-chip experiment to determine the respective regulons of RpoHI and RpoHII in Rhodobacter sphaeroides. We grew R. sphaeroides in aerobic conditions and induced either proteins ectopically and immuno-precipitated the regions of the genomic DNA interacting with the sigma factors. DNA immunoprecipitated with antibodies against RpoHI or RpoHII was labelled with Cy5 and hybridized on two-color tilling arrays (triplicates for each) with genomic DNA as an input control labelled with Cy3.

2012-08-02 | E-GEOD-39711 | biostudies-arrayexpress

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