Dataset Information

Comprehensive Identification of Phosphorylation Sites on the Numb Endocytic Adaptor Protein

ABSTRACT: Numb is an endocytic adaptor protein that functions in the endocytosis and intracellular trafficking of membrane receptors and adhesion molecules. Numb localization and function have been shown to be regulated by phosphorylation through atypcial protein kinase C at several key sites. Here, we use Liquid Chromatogrpahy, Tandem Mass Spectrometry to identify additional Numb phosphorylation sites not yet characterized.

INSTRUMENT(S): LTQ Orbitrap, LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Dr Jane McGlade

PROVIDER: MSV000080218 | MassIVE | Tue Oct 04 11:57:00 BST 2016

SECONDARY ACCESSION(S): PXD000997

REPOSITORIES: MassIVE

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Publications

Comprehensive identification of phosphorylation sites on the Numb endocytic adaptor protein.

Krieger Jonathan R JR Taylor Paul P Moran Michael F MF McGlade C Jane CJ

Proteomics 20150101 2-3

Numb is an adaptor protein that functions in the endocytosis and intracellular trafficking of membrane receptors and adhesion molecules. Previous studies have indicated that Numb localization and function are regulated through phosphorylation by atypical protein kinase C at several key sites. Here, using LC-MS/MS, we report the identification of 25 serine/threonine Numb phosphorylation sites, and a single tyrosine phosphorylation site. Amino acid sequences flanking several of the sites identifie ...[more]

PMID: 25403733

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Project description:Background: Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and elucidating the mechanisms underlying ccRCC growth and progression will provide clues to treat this aggressive malignant tumor. Method: Expression studies of RING ligase praja2 and tyrosine kinase receptors (RTKs) in renal cell carcinoma was evaluated by immunoblot and immunohistochemistry. Gene transcription profiling in RCC cells was evaluated by RNA sequencing and Ingenuity Pathway Analysis. Imunofluorescence assays were used to evaluate the intracellular distribution and clearance of membrane receptors in cells devoid of praja2. Ubiquitin modifications and protein-protein interaction networks were monitored by IP, western blot, mass spectrometry and proteomic analysis. Experiments in vitro and in vivo were performed to define the role of praja2 in RTKs turnover, metabolism, renal cell carcinoma growth and metastatic diffusion. Results: We report here that the ubiquitin ligase praja2 controls endocytosis and the signal strength of different types of receptors (EGFR, VEGFR, TfR). Praja2 forms a complex with- and ubiquitinates the adaptor protein AP2m required for the activity of ATPase and acidification of endosomes and lysosomes necessary for receptor endocytosis and clearance. Downregulation of praja2 blocks the endocytosis of several membrane receptors, including EGFR, VEGFR and TfR and amplifies mitogenic and proliferative signaling as shown in the clear cell renal cell carcinoma (ccRCC). Restoring praja2 expression in RCC cells reduces EGFR levels, rewires cancer cell metabolism and inhibits growth and metastatic diffusion. In vivo, praja2 knockout mice show upregulation of tyrosine kinase receptors (RTKs) and pronounced histopathological renal alterations. Conclusion: Our findings identify praja2 as an important regulator of tyrosine kinase receptor(s) turnover and can be considered a bona fide oncosuppressor because it finely regulates signaling of several types of receptors and its loss supports kidney cancer growth and diffusion.

Dataset Information

Comprehensive Identification of Phosphorylation Sites on the Numb Endocytic Adaptor Protein

Publications

Comprehensive identification of phosphorylation sites on the Numb endocytic adaptor protein.

Similar Datasets

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