Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Analysis of PKD1 autophosphorylation in vitro and confirmation of stoichiometric S742 phosphorylation of a recombinant PRKD1 kinase domain

ABSTRACT: Protein kinase D1 (PRKD1, also referred to as PKD1), has been proposed to undergo autophosphorylation on serine 742 and potentially also on serine 738 in the activation loop of its kinase domain. We used recombinant PKD1 kinase domain to test whether PKD1 is indeed able to undergo autophosphorylation of its kinase domain and how many sites could be phosphorylated. In order to obtain fully phosphorylated PKD1 kinase domain we adapted our autophosphorylation protocol to obtain monophosphorylated PKD1 kinase domain and determined the phosphorylation site by ArgC digest and phosphopeptide mapping.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Markus Hartl

LAB HEAD: Thomas A. Leonard

PROVIDER: PXD013216 | Pride | 2019-08-19

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Publications

A ubiquitin-like domain controls protein kinase D dimerization and activation by trans-autophosphorylation.

Elsner Daniel J DJ Siess Katharina M KM Gossenreiter Thomas T Hartl Markus M Leonard Thomas A TA

The Journal of biological chemistry 20190812 39

Protein kinase D (PKD) is an essential Ser/Thr kinase in animals and controls a variety of diverse cellular functions, including vesicle trafficking and mitogenesis. PKD is activated by recruitment to membranes containing the lipid second messenger diacylglycerol (DAG) and subsequent phosphorylation of its activation loop. Here, we report the crystal structure of the PKD N terminus at 2.2 Å resolution containing a previously unannotated ubiquitin-like domain (ULD), which serves as a dimerization ...[more]

PMID: 31406020

Similar Datasets

Cross-link mass spectrometry reveals conformational changes within the N lobe and the activation loop of PDK1 upon dimerization

Project description:Cross-linking mass spectrometry analysis of the PDK1 kinase domain dimer that undergoes trans-autophosphorylation.

2022-05-20 | PXD031827 | Pride

Substrate phosphorylation of protein microarray by leucine-rich repeat kinase 2

Project description:Numerous leucine-rich repeat kinase 2 mutations identified throughout the protein are associated with Parkinson disease, however the activating G2019S kinase domain mutation is currently regarded as the most common cause of familial and sporadic forms of this disorder. Despite studies demonstrating the prominent role that its kinase activity plays in the pathobiology of leucine-rich repeat kinase 2, few substrates have been identified and only a subset of these have been linked to disease. Therefore, we utilized protein microarrays to screen over 9,000 human proteins in an unbiased radiometric assay for potential targets of the kinase. ProtoArrayM-bM-^DM-" Human Protein Microarrays v5.0 (Invitrogen, Carlsbad, CA, USA) were used following the manufactureM-bM-^@M-^Ys protocol (ProtoArray Kinase Substrate Identification Kit). Briefly, slides were equilibrated at 4C for 15 min before blocking in 1% BSA in PBS for 1 h at 4oC with gentle shaking. Recombinant G2019S or D1994A glutathione-S-transferase (GST)-LRRK2 (970-2527) (Invitrogen) was diluted to 50nM in 20mM Tris (pH 7.5), 10mM MgCl2, 1mM EGTA, 1mM Na3VO4, 5mM beta-glycerophosphate, 2mM DTT, 0.02% polysorbate 20, and 10 mCi /mL of [gamma- 33P]ATP (33 nM final concentration) in a total volume of 120uL. Slides were overlayed with buffer alone, or buffer containing G2019S or D1994A LRRK2, then covered with a coverslip and placed in a 50 mL conical tube for 1 h at 30oC. Afterwards, slides were washed with 0.5% SDS buffer and water followed by centrifugation. Dried slides were exposed to a PhosphorImager plate (Amersham Biosciences, Piscataway, NJ, USA), and scanned on a Storm 840 (Molecular Dynamics, Inc., Sunnyvale, CA, USA) at 50 microns.

2012-07-13 | E-GEOD-30496 | biostudies-arrayexpress

Mapping of phosphorylation sites on Sgk3 by LC-MS/MS

Project description:The phosphorylation profile of Sgk3 protein expressed either in Sf9 insect cells or HEK 293T human cells was assessed by mass spectrometry.

2022-02-17 | PXD026281 | Pride

PKD autoinhibition in trans prevents activation loop autophosphorylation in cis

Project description:HDX-MS analysis of the kinase domain of PKD1, showing that it forms an inactive dimer

2023-02-22 | PXD033139 | Pride

Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation

Project description:Pyk2 is a multidomain non-receptor tyrosine kinase that undergoes a complex, multi-stage activation process. Ca2+-flux induces conformational rearrangements that relieve autoinhibitory FERM domain interactions. The kinase domain phosphorylates a key linker residue to recruit Src kinase. Pyk2 and Src mutually phosphorylate activation loop residues to confer full activation. While the mechanisms of autoinhibition are established, the conformational dynamics associated with autophosphorylation and Src recruitment remain unclear. Here, we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map the conformational changes associated with Src-mediated activation segment phosphorylation in a Pyk2 construct encompassing FERM and kinase domains (residues 20-692). Results reveal increased dynamics at regulatory interfaces spanning FERM and kinase domains. Phosphorylation of the activation segment stabilizes two antiparallel beta strands linking activation and catalytic loops. Increased dynamics of the C-terminal end of the activation loop propagate to the F-helix, explaining how phosphorylation prevents reversion to the autoinhibitory FERM interaction. HDX-guided site-directed mutagenesis and kinase activity profiling establish a mechanism for phosphorylation-induced active site sculpting to confer high activity.

2023-03-08 | PXD034028 | Pride

Cyclin dependent kinase 5 (CDK5) regulates the circadian clock

Project description:We show that the cyclin-dependent kinase 5 (CDK5) regulates the mammalian circadian clock via phosphorylation of PER2. CDK5 phosphorylated PER2 at serine residue 394 (S394) as shown by an in vitro kinase assay.

2019-12-04 | PXD012068 | Pride

Identify HILPS-interacting proteins and HIF1a phosphorylation sites

Project description:To dissect the molecular mechanisms of HILPS in promoting cell survival under hypoxia, we conducted RNA pull-down assay in combination with mass spectrometry analysis to identify HILPS-interacting proteins. Pull-down of antisense HILPS was used as a negative control. To identify the potential phosphorylation site(s), we conducted the in vitro kinase assay in combination with mass spectrometry analysis using PLK1-T210D, recombinant GST-HIF1a and cold ATP. A kinase assay using PLK1-K82R was included as a control.

2023-10-19 | PXD042226 | Pride

Determination and Inference of Eukaryotic Transcription Factor Sequence Specificity

Project description:The DNA sequence preferences of the vast majority of eukaryotic transcription factors (TFs) are unknown. Using an approach designed to broadly sample both DNA-binding domain types and eukaryotic clades, we have determined DNA-binding motifs for 1,033 TFs from 131 diverse eukaryotes, encompassing 54 domain types. Closely related orthologs and paralogs typically have very similar sequence preferences; this property allows inference of motifs for roughly one third of the 166,851 known or predicted eukaryotic TFs. While the origins of most motifs can be dated to hundreds of millions of years ago, we also characterize more recent TF expansions. Sequences matching the motifs are enriched upstream of TSS in most eukaryotic lineages, and at informative eQTL SNPs in Arabidopsis promoters, demonstrating their utility in mapping transcriptional networks. The motifs are housed at http://cisbp.ccbr.utoronto.ca Protein binding microarray (PBM) experiments were performed for a set of 1048 diverse eukaryotic transcription factors. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. Details of the PBM protocol are described in Berger et al., Nature Biotechnology 2006.

2014-08-01 | E-GEOD-53348 | biostudies-arrayexpress

Substrate selectivity for semisynthetic CK2 proteins with various posttranslational modifications

Project description:Protein Ser/Thr kinase CK2 is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. The CK2 catalytic subunit, CK2α, is modified by O-GlcNAc on Ser347 proximal to a Cdk1 phosphorylation site at Thr344 on the same protein. The substrate selectivity for protein kinase CK2 was examined by performing kinase assays on protein microarrays spotted with 17,000 human proteins. Semisynthetic CK2α proteins were prepared to contain an unmodified C-terminal tail, S-GlcNAc-Serine at S347, or Pfa (non-hyrdolyzeable phosphomimic) at T344. These semisynthetic proteins were used to determine if the posttranslational modifications on CK2 alpha modulate the substrate selectivity for this pleiotropic kinase. The different semisynthetic CK2α proteins were tested alone and in the presence of the regulatory subunit CK2β since it is known that the CK2α subunit is active both in its isolated state and in the heterotetrameric state formed in the presence of the regulatory beta subunit. The CK2β subunit has been shown to modulate CK2 activity with some substrates and not others. In the study presented here, kinase assays were performed using three different semisynthetic CK2 alpha proteins: unmodified C-terminal tail; S-GlcNAc-Ser at 347; and Pfa (phosphomimic) at 344. The semisynthetic proteins were each tested alone and in the presence of the regualatory CK2 beta subunit. There were six different kinase conditions and each condition was performed in duplicate and one no kinase control was performed to eliminate autophorylated proteins.

2011-10-24 | E-GEOD-33149 | biostudies-arrayexpress

Project description:We found that LC3B is phosphorylated in vitro by STK3, STK4, NEK9, and PKCζ. To identify phosphorylation sites following in vitro phosphorylation, GST-LC3B was overexpressed in E. coli, purified using glutathione-Sepharose 4 Fast Flow beads and incubated in kinase assays with recombinant kinases or FLAG-tagged kinases immunopurified from transiently transfected HEK293 cells

2020-01-23 | PXD016681 | 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