PXD031988

PXD031988 is an original dataset announced via ProteomeXchange.

Title	Flotillin-2 regulates EGFR activation, degradation, and cancer growth
Description	Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in a variety of cancers. The ubiquitin ligase Cbl regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes in the absence and presence of EGF stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacts with both Cbl and EGFR. Downregulation of EGFR upon FLOT2 loss is Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Stable overexpression of FLOT2 in HeLa cells decreased EGF-stimulated EGFR phosphorylation and ubiquitination. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induces EGFR-dependent proliferation and anchorage-independent cell growth. Lastly, FLOT2 knockout increases tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrate that FLOT2 negatively regulates EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation. FLOT2 negatively regulates proliferation in vitro and in vivo.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_08:41:10.160.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Xu Zhang
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2022-03-02 01:12:49	ID requested
1	2023-03-11 02:54:40	announced
⏵ 2	2023-11-14 08:41:12	announced	2023-11-14: Updated project metadata.

Wisniewski DJ, Liyasova MS, Korrapati S, Zhang X, Ratnayake S, Chen Q, Gilbert SF, Catalano A, Voeller D, Meerzaman D, Guha U, Porat-Shliom N, Annunziata CM, Lipkowitz S, Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth. J Biol Chem, 299(1):102766(2023) [pubmed]

submitter keyword: mass spectrometry, phosphorylation, MAPK, cell proliferation, tumor growth,Cbl, EGFR, FLOT2

Stanley Lipkowitz
contact affiliation	Women’s Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892
contact email	lipkowis@navmed.nci.nih.gov
lab head
Xu Zhang
contact affiliation	NIH/NCI
contact email	xu.zhang@nih.gov
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD031988
     1. Label: PRIDE project
     2. Name: Flotillin-2 regulates EGFR activation, degradation, and cancer growth