RPXD034752

RPXD034752 is container for one or more analyses. The general container metadata is provided below and the table under Dataset History provides links to the various reanalyses have been provided (if any) in this container.

Title	JPST000150-4 in vitro kinase reaction, ERK1 [Reanalysis: JPST000150]
Description	Protein kinase selectivity is largely governed by direct binding to the target site(s) on the substrate. Thus, substrate determinants identified from sequences around phosphorylation sites are desirable resources for matching kinases to their substrates. In this study, we tried to identify kinase-selective substrate determinants, including motif sequences, based on large-scale discovery of kinase/substrate pairs. For this purpose, we employed a combination strategy of in vitro kinase reaction followed by LC−MS/MS analysis and applied it to three well-studied kinases: c-AMP regulated protein kinase A (PKA), extracellular signal-regulated kinase 1 (ERK1), and RAC-alpha serine/threonine-protein kinase (AKT1). Cellular proteins were fractionated, dephosphorylated with thermosensitive alkaline phosphatase, phosphorylated with the target kinase, and digested with Lys-C/trypsin, and then phosphopeptides were enriched using TiO2-based hydroxy acid-modified metal oxide chromatography (HAMMOC) and subjected to LC−MS/MS. As a result, 3585, 4347, and 1778 in vitro phosphorylation sites were identified for PKA, ERK1, and AKT1, respectively. As expected, these extensive identifications of phosphorylation sites enabled extraction of both known and novel motif sequences, and this in turn permitted fine discrimination of the specificities of PKA and AKT1, which both belong to the AGC kinase family. Other unique features of the kinases were also characterized, including phospho-acceptor preference (Ser or Thr) and bias ratio of singly/multiply phosphorylated peptides. More motifs were found with this methodology as compared with target kinase phosphorylation of peptides obtained by predigestion of proteins with Lys-C/trypsin. Thus, this approach to characterization of kinase substrate determinants is effective for identification of kinases associated with particular phosphorylation sites. [Original project description]
HostingRepository	jPOST
AnnounceDate	2023-04-01
AnnouncementXML	Submission_2023-04-01_01:00:44.616.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/RPXD034752
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Reanalysis of JPST000150
RepositorySupport	Supported dataset by repository
PrimarySubmitter	jPOST reanalysis
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide; O-phospho-L-serine; O-phospho-L-threonine; O4'-phospho-L-tyrosine
Instrument	TripleTOF 5600

Reanalysis	Revision	Datetime	Status	Title / ChangeLog Entry
	0	2022-06-20 17:57:59	ID requested
0	1	2023-04-01 01:00:44	announced	JPST000150-4 in vitro kinase reaction, ERK1 [Reanalysis: JPST000150]

no publication

submitter keyword: reanalysis, phosphoproteome, kinase/substrate, motif sequence, phosphoacceptor, multiply phosphorylated peptide, ERK1

Yasushi Ishihama
lab head
jPOST reanalysis
contact affiliation	jPOST
dataset submitter

jPOST dataset URI

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.jpostdb.org/JPST001756/