Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells.
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.
Iwama
A
A
Department of Cell Differentiation, Kumamoto University School of Medicine, Japan.
Okano
K
K
Sudo
T
T
Matsuda
Y
Y
Suda
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D006412:Hematopoietic Stem Cells / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics*; D011518:Proto-Oncogene Proteins; D019859:Proto-Oncogene Proteins c-met; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D051941:fms-Like Tyrosine Kinase 3
D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; C497971:Flt3 protein, mouse; D011505:Protein-Tyrosine Kinases; D019859:Proto-Oncogene Proteins c-met; D020794:Receptor Protein-Tyrosine Kinases; D051941:fms-Like Tyrosine Kinase 3