PXD048758

PXD048758 is an original dataset announced via ProteomeXchange.

Title	SKAP2 acts downstream of CD11b/CD18 and regulates neutrophil effector function
Description	Background: The importance of CD11b/CD18 expression in neutrophil effector functions is well known. Beyond KINDLIN3 and TALIN1, which are involved in the induction of the high-affinity binding CD11b/CD18 conformation, the signaling pathways that orchestrate this response remain incompletely understood. Method: We performed an unbiased screening method for protein selection by biotin identification (BioID) and investigated the KINDLIN3 interactome. We used liquid chromatography with tandem mass spectrometry as a powerful analytical tool. Generation of NB4 CD18, KINDLIN3 or SKAP2 knock out neutrophils was achieved using CRISPR-Cas9 technology and the cells were examined for their effector function using flow cytometry, live cell imaging, microscopy, adhesion or antibody-dependent cellular cytotoxicity (ADCC). Results: Among the 325 proteins significantly enriched, we identified Src Kinase-Associated Phosphoprotein2 (SKAP2), a protein involved in actin polymerization and integrin-mediated outside-in signaling. CD18 immunoprecipitation in primary or NB4 neutrophils demonstrated the presence of SKAP2 in the CD11b/CD18 complex at steady state. Under this condition, adhesion to plastic, ICAM-1 or fibronectin was observed in the absence of SKAP2, which could be abrogated by blocking the actin-rearrangements with latrunculin B. Upon stimulation of SKAP2-deficient neutrophils, adhesion to fibronectin was enhanced whereas CD18 clustering was strongly reduced. This response corresponded with significantly impaired CD11b/CD18-dependent NADPH oxidase activity, phagocytosis and cytotoxicity against tumor cells. Conclusion: Our results suggest that SKAP2 has a dual role. It may restrict CD11b/CD18-mediated adhesion only under resting conditions, but its major contribution lies in the regulation of dynamic CD11b/CD18-mediated actin-rearrangements and clustering as required for cellular effector functions of human neutrophils.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_06:30:45.490.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Floris van Alphen
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	iodoacetamide derivatized residue
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2024-01-22 03:58:52	ID requested
1	2024-02-16 02:02:11	announced
⏵ 2	2024-10-22 06:30:45	announced	2024-10-22: Updated project metadata.

10.3389/FIMMU.2024.1344761;

submitter keyword: phagocytosis, Src Kinase Associated Phosphoprotein 2 (SKAP2), CD11b/CD18 integrin, filamentous actin, antibody-dependent cellular cytotoxicity (ADCC), adhesion,neutrophils

Hanke L. Matlung
contact affiliation	Dept. of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands, and Landsteiner Laboratory, University of Amsterdam, Amsterdam, the Netherlands
contact email	h.matlung@sanquin.nl
lab head
Floris van Alphen
contact affiliation	Sanquin Research
contact email	F.vanalphen@sanquin.nl
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD048758
     1. Label: PRIDE project
     2. Name: SKAP2 acts downstream of CD11b/CD18 and regulates neutrophil effector function