title: Identification and selected reaction monitoring (SRM) quantification of endocytosis factors associated with Numb.
abstract: Numb is an endocytic adaptor protein that regulates the endocytosis and trafficking of transmembrane receptors including Notch, E-cadherin, and integrins. Vertebrate Numb is alternatively spliced at exons 3 and 9 to give rise to four protein isoforms. Expression of these isoforms varies at different developmental stages, and although the function of Numb isoforms containing exon 3 has been studied, the role of exon 9 inclusion has not been shown. Here we use affinity purification and tandem mass spectrometry to identify Numb associated proteins, including novel interactions with REPS1, BMP2K, and BCR. In vitro binding measurements indicated exon 9-independent Numb interaction with REPS1 and Eps15 EH domains. Selected reaction monitoring mass spectrometry was used to quantitatively compare the proteins associated with the p72 and p66 Numb isoforms, which differ by the exon 9 region. This showed that significantly more EPS15 and three AP-2 subunit proteins bound Numb isoforms containing exon 9. The EPS15 preference for exon 9-containing Numb was confirmed in intact cells by using a proximity ligation assay. Finally, we used multiplexed selected reaction monitoring mass spectrometry to assess the dynamic regulation of Numb association with endocytic proteins. Numb hyper-phosphorylation resulted in disassociation of Numb endocytic complexes, while inhibition of endocytosis did not alter Numb association with the AP-2 complex but altered recruitment of EPS15, REPS1, and BMP2K. Hence, quantitative mass spectrometric analysis of Numb protein-protein interactions has provided new insights into the assembly and regulation of protein complexes important in development and cancer.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5G 2M9, Canada.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Krieger
authors.forename: Paul
authors.initials: P
authors.lastname: Taylor
authors.forename: Aaron S
authors.initials: AS
authors.lastname: Gajadhar
authors.forename: Abhijit
authors.initials: A
authors.lastname: Guha
authors.forename: Michael F
authors.initials: MF
authors.lastname: Moran
authors.forename: C Jane
authors.initials: CJ
authors.lastname: McGlade
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002853:Chromatography, Liquid; D004705:Endocytosis / Q000235:genetics*; D005091:Exons; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051562:Proto-Oncogene Proteins c-bcr / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry / Q000379:methods; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; C486206:EPS15 protein, human; C436492:KLF12 protein, human; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C106293:Numb protein, mouse; D020033:Protein Isoforms; C484665:REPS1 protein, human; C496613:BCR protein, human; D051562:Proto-Oncogene Proteins c-bcr