Iwama A, Okano K, Sudo T, Matsuda Y, Suda T
Department of Cell Differentiation, Kumamoto University School of Medicine, Japan.
Blood. 1994 Jun 1;83(11):3160-9.
To identify the novel receptor tyrosine kinases (RTKs) critical to the proliferation of hematopoietic stem cells, we performed polymerase chain reaction-based cloning from highly purified murine hematopoietic stem cells. Lineage marker-negative, c-KIT-positive, and Ly6A/E- or Sca-1-positive (Lin-c-KIT+Sca-1+) cells were sorted by a fluorescence-activated cell sorter. Two sets of degenerate oligonucleotide primers were directed to the conserved sequences of the catalytic domain, and were used to amplify cDNAs that encode protein tyrosine kinases (PTKs). One hundred cDNA clones were sequenced and 8 RTKs were identified, as well as 12 non-RTKs and 2 serine/threonine kinases. Sixteen cDNAs were identical to the known kinase genes (PKC beta, JAK-1, JAK-2, TYK-2, HCK, FGR, FYN, BLK, c-FES, FER, c-ABL, c-KIT, FLK-1, FLK-2, IGF1R, and ECK). Six novel cDNA sequences (stk series) were identified. However, three of them turned out to be BPK, RYK, and TEK. The remaining three showed high homology to S6 kinase II, JAK-2, and v-SEA/c-MET, respectively. Characterization of full-length cDNA sequence of the v-SEA/cMET-related gene showed that this was a novel RTK gene and we named this gene STK (stem cell-derived tyrosine kinase). We identified two distinct forms of STK cDNA; the short one encoded a putative truncated protein that lacked most of the extracellular domain. STK was expressed at various stages of hematopoietic cells, including stem cells, but we could not detect any apparent expression in other adult tissues. The expression of the truncated form of mRNA was more predominant than that of the complete form. STK was assigned by fluorescent in situ hybridization to the R-positive F1 band of chromosome 9, the same region to which hepatic growth factor-like protein has been assigned. Characterization of these PTKs, including STK, will be helpful to elucidate the molecular mechanism of the growth regulation of hematopoietic stem cells.
为了鉴定对造血干细胞增殖至关重要的新型受体酪氨酸激酶（RTK），我们从高度纯化的小鼠造血干细胞中进行了基于聚合酶链反应的克隆。通过荧光激活细胞分选仪分选谱系标志物阴性、c-KIT阳性以及Ly6A/E或Sca-1阳性（Lin-c-KIT+Sca-1+）的细胞。两组简并寡核苷酸引物针对催化结构域的保守序列，用于扩增编码蛋白酪氨酸激酶（PTK）的cDNA。对100个cDNA克隆进行测序，鉴定出8种RTK、12种非RTK和2种丝氨酸/苏氨酸激酶。16个cDNA与已知激酶基因（PKCβ、JAK-1、JAK-2、TYK-2、HCK、FGR、FYN、BLK、c-FES、FER、c-ABL、c-KIT、FLK-1、FLK-2、IGF1R和ECK）相同。鉴定出6个新的cDNA序列（stk系列）。然而，其中3个结果是BPK、RYK和TEK。其余3个分别与S6激酶II、JAK-2和v-SEA/c-MET具有高度同源性。对v-SEA/cMET相关基因全长cDNA序列的表征表明，这是一个新的RTK基因，我们将该基因命名为STK（干细胞衍生的酪氨酸激酶）。我们鉴定出两种不同形式的STK cDNA；短的一种编码一种推定的截短蛋白，该蛋白缺乏大部分细胞外结构域。STK在造血细胞的各个阶段表达，包括干细胞，但在其他成年组织中未检测到任何明显表达。截短形式的mRNA表达比完整形式更占优势。通过荧光原位杂交将STK定位于9号染色体的R阳性F1带，肝生长因子样蛋白也定位于该区域。对包括STK在内的这些PTK的表征将有助于阐明造血干细胞生长调节的分子机制。
