Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells

Abstract

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.

Restricted expression of the ron gene encoding the macrophage stimulating protein receptor during mouse development

“…Several hybridizing cDNA clones were isolated and sequenced. The sequence of the longest of these cDNAs (2,100 bp) covered nucleotides 2,634-4,720 on the full-length mouse ron cDNA sequence reported recently by Iwama et al (1994). A 1.0 kb murine ron cDNA fragment (nucleotides 2,634-3,659) was used to generate [36Sl-labelled antisense riboprobes, which were used for in situ hybridization experiments on serial sections of mouse embryos and fetuses at each gestational day from 8.5 to 18.5 days post-coitum (dpc), as well as on newborn specimens.…”

“…A mouse skin cDNA library in XZAP-XR was purchased from Stratagene (La Jolla, CA) and screened under low stringency conditions with a 1 kbp human ron cDNA probe encompassing the sequences coding for the tyrosine kinase domain, the transmembrane region, and part of the extracellular domains (nucleotides 2,461 to 3,444, Ronsin et al, 1993). Four hundred thousand plaques were screened, allowing isolation of 3 positive clones, the longest corresponding to nucleotides 2,6344,720 of the published mouse sequence (Iwama et al, 1994).…”

Receptor tyrosine kinases and the regulation of macrophage activation

“…In contrast, the Axl/Tyro3/Mer family of receptors [5,6] contains two Ig-like domains and two fibronectin type III repeats in the ectodomain and a contiguous kinase domain with two tandem tyrosines in the activation loop. The murine STK/human RON receptor [7,8], a member of the MET family of RTKs, is closely related to the Tyro3 family and shares a similar kinase structure. The ectodomain of STK, however, is composed of a disulfide-linked extracellular ␣ chain and transmembrane ␤ chain, the structure of which is poorly characterized.…”

Growth Factor Induction of Cytosolic Protein Tyrosine Kinase Activity in Human Haemopoietic Progenitor Cells Isolated by Flow Cytometry

“…In addition to c-kit and flk-2, RNA encoding for a number of tyrosine kinases has been identified in haemopoietic progenitor cells. In a recent study, Iwama et al (1994) identified genetic material coding for 20 receptor and nonreceptor tyrosine kinases using reverse transcriptase polymerase chain reaction to analyse RNA of lineage negative, c-kit + murine haemopoietic stem cells. That study identified a novel tyrosine kinase gene, STK, as a member of a subfamily of receptor tyrosine kinase genes that included c-MET, c-SEA and the human homologue of STK, RON.…”