Comprehensive identification of phosphorylation sites on the Numb endocytic adaptor protein.
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 identified conform to consensus motifs for cyclin-dependent kinase 5 (CDK5). In vitro kinase assays and immunoblotting confirmed that CDK5 phosphorylates Numb. LC-MS/MS analysis identified specific CDK5-directed phosphorylation of Numb at position S288 and at two additional regions. Therefore, Numb is likely to exist in multiple phospho-isoforms, and may be subject to phosphorylation-mediated regulation downstream of CDK5. These findings provide a basis for further investigations into the complex role of Numb phosphorylation in regulating its subcellular localization, protein interactions, and function. All MS data have been deposited in the ProteomeXchange with identifier PXD000997 (http://proteomecentral.proteomexchange.org/dataset/PXD000997).
Program in Cell Biology, The Hospital For Sick Children, Toronto, ON, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada; The Arthur and Sonia Labatt Brain Tumour Research Center, The Hospital for Sick Children, Toronto, ON, Canada.
Jonathan R
JR
Krieger
Paul
P
Taylor
Michael F
MF
Moran
C Jane
CJ
McGlade
D000595:Amino Acid Sequence; D051360:Cyclin-Dependent Kinase 5 / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000032:analysis / Q000378:metabolism
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C113026:NUMB protein, human; D020033:Protein Isoforms; D014443:Tyrosine; D051360:Cyclin-Dependent Kinase 5
Cyclin-dependent kinase 5; Mass spectrometry; Numb; Phosphorylation