PXD054542

PXD054542 is an original dataset announced via ProteomeXchange.

Title	LC/MS/MS identification of phosphor-sites of Human ATR-domain (330-616 aa) expressed and purified from E. coli after in vitro phosphorylation.
Description	Pin1, a peptidyl-prolyl cis/trans isomerase (PPIase), and PP2A regulate ATRS428P429 isomerization, they act oppositely depending on the phosphorylation status of ATR(S428). In addition, whether the isomerization at the ATR-S428P429 motif along with the BH3 domain accessibility is exclusively responsible for the interaction between cis-ATR and tBid in a ATR kinase activity-independent manner may need further confirmation with purified proteins lacking the ATR kinase domain in vitro. To address these issues under fully controlled experimental conditions, plasmid vectors for expressing a N-terminally His-tagged 33 kDa fragment of ATR containing both the isomerization motif (WT, mutants S428A-P429 or S428-P429A) and BH3 domain (the fragment is referred to as the ATR-iBH3 domain) were constructed. The ATR-iBH3 domains with an isomerization motif of WT, S428A, or P429A were purified after overexpression of the corresponding constructs in E. coli. These ATR-iBH3 domain proteins do not contain the kinase domain of ATR, ensuring that any observed functions of ATR-iBH3 are independent of ATR’s kinase domain or its activity. The purified ATR-iBH3 domains were phosphorylated with CDK1 or mock treated, followed by treatment with purified tBid protein, and subsequently subjected to pull-down assays using Ni-NTA beads to isolate the His-tagged ATR-iBH3 domains and any associated tBid. As tBid was efficiently pulled down with both ATR-iBH3WT and ATR-iBH3S428A in the absence of CDK1, indicating their interaction with tBid. In contrast, little or no tBid was pulled down by ATR-iBH3P429A. These results align well with their isomerization states previously observed in full-length of ATR: cytosolic ATR-WT and ATR-S428A are in a similar cis-isomeric form, whereas ATR-P429A adopts a trans-isomeric form. The results also imply that the prolyl isomeric conformations of full-length ATR originate from the ATR-iBH3 domain. On the other hand, phosphorylation of the ATR-iBH3WT domain by CDK1 significantly diminished its interaction with tBid, while CDK1 treatment on the non-phosphorable ATR-iBH3S428A domain had no effect on its binding to tBid. There are two possible CDK1 recognition motifs, Ser428Pro429 and Thr464Ala465 in the 33 kDa domain. LC-MS/MS spectrometric analysis of the CDK1-treated ATR-iBH3WT protein identified p-S428 and indicated that the phosphorylation efficiency at S428 is 10 times higher than at T464. Moreover, even hypothetically considering phosphorylation at other residues, the lack of effect of the CDK1 treatment on the ATR-iBH3S428A domain’s binding to tBid suggests that the hypothetic phosphorylation would not affect the ATR-iBH3 domain’s interaction with tBid.
HostingRepository	PRIDE
AnnounceDate	2026-09-08
AnnouncementXML	Submission_2026-09-08_09:27:39.268.xml
DigitalObjectIdentifier	https://doi.org/10.6019/PXD054542
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Himadri Biswas
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: NEWT:562;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2024-08-02 09:28:19	ID requested
⏵ 1	2026-09-08 09:27:39	announced

10.6019/PXD054542;

submitter keyword: Biotin modification,ATR-domain, cis/trans prolyl isomerization, Mass protein footprinting, BH3-like domain

Himadri Biswas
contact affiliation	Himadri Biswas, Ph. D. Post-doctoral associate, Dept. of Cancer Biology, University of Toledo, Ohio, 43614
contact email	Himadri.Biswas@UToledo.Edu
lab head
Himadri Biswas
contact affiliation	University of Toledo
contact email	himadri.biswas@utoledo.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD054542
     1. Label: PRIDE project
     2. Name: LC/MS/MS identification of phosphor-sites of Human ATR-domain (330-616 aa) expressed and purified from E. coli after in vitro phosphorylation.