Boivin Dominique, Labbé David, Fontaine Nicolas, Lamy Sylvie, Beaulieu Edith, Gingras Denis, Béliveau Richard
Laboratoire de Médecine Moléculaire, Chemistry Department, Université du Québec à Montréal, Canada H3C 3P8.
Biochemistry. 2009 May 12;48(18):3998-4007. 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.
CD133（prominin-1）是一种跨膜糖蛋白，在正常干细胞、癌症干细胞、祖细胞、视杆光感受器细胞以及多种上皮细胞表面表达。尽管CD133被广泛用作各种体细胞和假定的癌症干细胞的标志物，但其对干细胞诸如自我更新和分化等基本特性的作用仍不清楚。CD133含有一个短的C末端胞质结构域，带有五个酪氨酸残基，包括一个尚未研究的共有酪氨酸磷酸化位点。在本研究中，我们表明CD133在人髓母细胞瘤D283和Daoy细胞中以Src家族激酶依赖性方式发生磷酸化。在过表达Src和Fyn酪氨酸激酶的Daoy细胞中以及使用重组蛋白在体外，CD133的胞质结构域发生酪氨酸磷酸化。缺失CD133的C末端胞质结构域可显著降低其被Src磷酸化的程度。为了鉴定CD133中的酪氨酸磷酸化位点，我们使用了基质辅助激光解吸/电离四极杆飞行时间质谱（MALDI Q-TOF）和液相色谱串联质谱（LC-MS/MS）。通过质谱分析酪氨酸磷酸化的CD133以及定点诱变确定，酪氨酸-828和非共有酪氨酸-852是体外和完整细胞中的主要酪氨酸磷酸化位点。鉴定出CD133是Src家族酪氨酸激酶的底物，这表明CD133的胞质结构域可能在其功能调节中起重要作用。
