The stem cell marker CD133 (prominin-1) is phosphorylated on cytoplasmic tyrosine-828 and tyrosine-852 by Src and Fyn tyrosine kinases.

Abstract

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.

• Authors: Dominique Boivin, David Labbé, Nicolas Fontaine, Sylvie Lamy, Edith Beaulieu, Denis Gingras, Richard Béliveau
• Journal: Biochemistry (Biochemistry) Vol. 48 Issue 18 Pg. 3998-4007 (May 12 2009) ISSN: 1520-4995 [Electronic] United States
• PMID: 19296573 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• AC133 Antigen
• Antigens, CD
• DNA Primers
• Glycoproteins
• PROM1 protein, human
• Peptides
• Tyrosine
• Proto-Oncogene Proteins c-fyn
• src-Family Kinases

Topics

• AC133 Antigen
• Amino Acid Sequence
• Antigens, CD (chemistry, genetics, metabolism)
• Base Sequence
• Chromatography, Liquid
• DNA Primers
• Glycoproteins (chemistry, genetics, metabolism)
• Molecular Sequence Data
• Mutagenesis, Site-Directed
• Peptides (chemistry, genetics, metabolism)
• Phosphorylation
• Polymerase Chain Reaction
• Proto-Oncogene Proteins c-fyn (metabolism)
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• Stem Cells (immunology)
• Tandem Mass Spectrometry
• Tyrosine (metabolism)
• src-Family Kinases (metabolism)