The stem cell marker CD133 (prominin-1) is phosphorylated on cytoplasmic tyrosine-828 and tyrosine-852 by Src and Fyn tyrosine kinases.
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.
Boivin
Dominique
D
Laboratoire de Médecine Moléculaire, Chemistry Department, Université du Québec à Montréal, Canada H3C 3P8.
Labbé
David
D
Fontaine
Nicolas
N
Lamy
Sylvie
S
Beaulieu
Edith
E
Gingras
Denis
D
Béliveau
Richard
R
D000071916:AC133 Antigen; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002853:Chromatography, Liquid; D017931:DNA Primers; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013234:Stem Cells / Q000276:immunology*; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D000071916:AC133 Antigen; D015703:Antigens, CD; D017931:DNA Primers; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; D014443:Tyrosine; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases