PXD024621

PXD024621 is an original dataset announced via ProteomeXchange.

Title	The effect of AKT and AMPK site-specific phosphorylation on TBC1D4 biological activity
Description	TBC1D4 is a 160 kDa multi-domain containing RabGAP (Rab GTPase-activating protein) and a downstream target of the insulin and contraction-activated kinases AKT and AMPK. While phosphorylation of TBC1D4 has been linked to GLUT4 translocation from storage vesicles (GSVs) to the cell surface, its impact on TBC1D4 enzymatic function is not well understood as previous studies mostly investigated the truncated GAP domain lacking the phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using the baculovirus system. Size exclusion chromatography and co-immunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of ~600 kDa. Moreover, full-length TBC1D4 displayed similar substrate specificity but had a markedly higher specific GAP activity towards Rab10 compared to the truncated GAP domain. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4. In vitro phosphorylation with purified kinases, stable isotope-labelled [γ-18O4]ATP and Michaelis-Menten kinetics identified Ser324 (KM ~6 µM) and Thr649. (KM ~25 µM) as preferential sites for AKT whereas four sites, Ser348, Ser577, Ser595 (KM ~10 µM), Ser711 (KM ~79 µM), and Ser764 were found to be preferred targets for AMPK. Phosphorylation of TBC1D4 by AKT or AMPK did not alter the intrinsic RabGAP activity but disrupted interaction with the insulin-regulated aminopeptidase (IRAP), a resident protein of GSV and implicated in GLUT4 traffic. These findings provide evidence that insulin and contraction may regulate TBC1D4 primarily through recruitment of the RabGAP to GLUT4 vesicles.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_05:32:33.082.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Stefan Lehr
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	phosphorylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2021-03-09 23:17:05	ID requested
1	2022-02-16 17:04:01	announced
⏵ 2	2024-10-22 05:32:40	announced	2024-10-22: Updated project metadata.

10.1016/j.jbc.2021.100637;

Eickelschulte S, Hartwig S, Leiser B, Lehr S, Joschko V, Chokkalingam M, Chadt A, Al-Hasani H, AKT/AMPK-mediated phosphorylation of TBC1D4 disrupts the interaction with insulin-regulated aminopeptidase. J Biol Chem, 296():100637(2021) [pubmed]

submitter keyword: LC-MSMS, TBC1D4,protein phosphorylation

Prof. Hadi Al-Hasani
contact affiliation	Prof. Dr. Hadi Al-Hasani Direktor Institut für Klinische Biochemie und Pathobiochemie Deutsches Diabetes-Zentrum (DDZ) Leibniz-Zentrum für Diabetes-Forschung an der Heinrich-Heine-Universität Düsseldorf Auf´m Hennekamp 65 40225 Düsseldorf
contact email	Hadi.Al-Hasani@ddz.de
lab head
Stefan Lehr
contact affiliation	German Diabetes Center Institute for Clinical Biochemistry and Pathobiochemistry
contact email	stefan.lehr@ddz.de
dataset submitter

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.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024621

PRIDE project URI

• PRIDE
  1. PXD024621
     1. Label: PRIDE project
     2. Name: The effect of AKT and AMPK site-specific phosphorylation on TBC1D4 biological activity