PXD034028-1

PXD034028 is an original dataset announced via ProteomeXchange.

Title	Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation
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.
HostingRepository	PRIDE
AnnounceDate	2023-03-08
AnnouncementXML	Submission_2023-03-08_14:55:09.748.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	EricUnderbakke
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	Synapt MS

Revision	Datetime	Status	ChangeLog Entry
0	2022-05-23 03:40:16	ID requested
⏵ 1	2023-03-08 14:55:10	announced
2	2023-11-14 08:56:26	announced	2023-11-14: Updated project metadata.

submitter keyword: kinase, phosphorylation, HDX-MS

Eric StevenUnderbakke
contact affiliation	Roy J. Carver Dept. of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA USA
contact email	esu@iastate.edu
lab head
EricUnderbakke
contact affiliation	Iowa State University
contact email	esu@iastate.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD034028
     1. Label: PRIDE project
     2. Name: Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation