The Stem Cell Marker CD133 (Prominin-1) is Phosphorylated on Cytoplasmic Tyrosine-828 and Tyrosine-852 by Src and Fyn Tyrosine Kinases

• Published in: Biochemistry (Easton) Vol. 48; no. 18; pp. 3998 - 4007
• Main Authors: Boivin, Dominique, Labbé, David, Fontaine, Nicolas, Lamy, Sylvie, Beaulieu, Édith, Gingras, Denis, Béliveau, Richard
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 12.05.2009
• Subjects: AC133 Antigen; Amino Acid Sequence; Antigens, CD - chemistry; Antigens, CD - genetics; Antigens, CD - metabolism; Base Sequence; Chromatography, Liquid; DNA Primers; Glycoproteins - chemistry; Glycoproteins - genetics; Glycoproteins - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Phosphorylation; Polymerase Chain Reaction; Proto-Oncogene Proteins c-fyn - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; src-Family Kinases - metabolism; Stem Cells - immunology; Tandem Mass Spectrometry; Tyrosine - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi900159d

CD133 (prominin-1) is a transmembrane glycoprotein expressed at the surface of normal and cancer stem cells, progenitor cells, rod photoreceptor cells, and a variety of epithelial cells. Although CD133 is widely used as a marker of various somatic and putative cancer stem cells, its contribution to fundamental properties of stem cells such as self-renewal and differentiation remains unknown. CD133 contains a short C-terminal cytoplasmic domain with five tyrosine residues, including a consensus tyrosine phosphorylation site that has not yet been investigated. In this study, we show that CD133 is phosphorylated in human medulloblastoma D283 and Daoy cells, in a Src family kinase-dependent manner. The cytoplasmic domain of CD133 is tyrosine phosphorylated in Daoy cells overexpressing Src and Fyn tyrosine kinases, as well as in vitro using recombinant proteins. Deletion of the C-terminal cytoplasmic domain of CD133 considerably reduced its phosphorylation by Src. To identify the tyrosine phosphorylation sites in CD133, we used matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI Q-TOF) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of tyrosine-phosphorylated CD133 by mass spectrometry and site-directed mutagenesis identified tyrosine-828 and the nonconsensus tyrosine-852 as the major tyrosine phosphorylation sites both in vitro and in intact cells. Identification of CD133 as a substrate for Src-family tyrosine kinases suggests that the cytoplasmic domain of CD133 might play an important role in the regulation of its functions.