Molecular Cloning and Characterization of Mouse TIE and TEK Receptor Tyrosine Kinase Genes and Their Expression in Hematopoietic Stem Cells

Abstract A receptor tyrosine kinase (RTK), TIE (tyrosine kinase that contains immunoglobulin-like loops and epidermal growth factor [EGF] homology domains), is expressed in vascular endothelial and hematopoietic cells. We generated monoclonal antibodies (MoAbs) against the extracellular domain of TIE and a polyclonal antibody against the TIE carboxyterminus and used them to analyze expression of TIE in hematopoietic cells. Western blotting detected two forms of TIE protein with a molecular mass of 135 and 130 kD in hematopoietic and endothelial cells. Northern blotting analysis revealed that TIE was expressed preferentially in undifferentiated cell lines, especially when megakaryocytic, but not erythroid differentiation was induced. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that TIE was predominantly expressed in the human hematopoietic progenitor fraction, CD34+ cells. Fluorescence- activated cell sorting (FACS) showed that 42% of CD34+ and 17% of KIT- positive (KIT+) cells were TIE-positive (TIE+). The majority (81%) of the primitive hematopoietic stem cells, CD34+CD38- cells, were TIE+. Assays of progenitor cells and long-term culture-initiating cells (LTC- IC) showed that the TIE+ fraction contained more primitive cells than the TIE- fraction. Some TIE+ cells were in the CD34- fraction, which were CD19+ and CD20+ (B cells). These findings indicate that TIE has a unique spectrum of expression in primitive hematopoietic stem cells and B cells. Although its ligand has not been identified, TIE and its ligand may establish a novel regulatory pathway not only in early hematopoiesis, but also in the differentiation and/or proliferation of B cells.

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

Blood ◽

10.1182/blood.v83.11.3160.bloodjournal83113160 ◽

1994 ◽

Vol 83 (11) ◽

pp. 3160-3169 ◽

Cited By ~ 5

Author(s):

A Iwama ◽

K Okano ◽

T Sudo ◽

Y Matsuda ◽

T Suda

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Hematopoietic Stem Cells ◽

Tyrosine Kinases ◽

Growth Regulation ◽

Chromosome 9 ◽

Cdna Clones ◽

Hematopoietic Stem ◽

Degenerate Oligonucleotide

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.

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

Blood ◽

10.1182/blood.v83.11.3160.3160 ◽

1994 ◽

Vol 83 (11) ◽

pp. 3160-3169 ◽

Cited By ~ 100

Author(s):

A Iwama ◽

K Okano ◽

T Sudo ◽

Y Matsuda ◽

T Suda

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Hematopoietic Stem Cells ◽

Tyrosine Kinases ◽

Growth Regulation ◽

Chromosome 9 ◽

Cdna Clones ◽

Hematopoietic Stem ◽

Degenerate Oligonucleotide

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.

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Predominant expression of a receptor tyrosine kinase, TIE, in hematopoietic stem cells and B cells

Blood ◽

10.1182/blood.v87.1.93.bloodjournal87193 ◽

1996 ◽

Vol 87 (1) ◽

pp. 93-101 ◽

Cited By ~ 2

Author(s):

M Hashiyama ◽

A Iwama ◽

K Ohshiro ◽

K Kurozumi ◽

K Yasunaga ◽

...

Keyword(s):

Stem Cells ◽

B Cells ◽

Tyrosine Kinase ◽

Hematopoietic Stem Cells ◽

Receptor Tyrosine Kinase ◽

Hematopoietic Cells ◽

Erythroid Differentiation ◽

Northern Blotting ◽

Hematopoietic Stem ◽

Epidermal Growth

A receptor tyrosine kinase (RTK), TIE (tyrosine kinase that contains immunoglobulin-like loops and epidermal growth factor [EGF] homology domains), is expressed in vascular endothelial and hematopoietic cells. We generated monoclonal antibodies (MoAbs) against the extracellular domain of TIE and a polyclonal antibody against the TIE carboxyterminus and used them to analyze expression of TIE in hematopoietic cells. Western blotting detected two forms of TIE protein with a molecular mass of 135 and 130 kD in hematopoietic and endothelial cells. Northern blotting analysis revealed that TIE was expressed preferentially in undifferentiated cell lines, especially when megakaryocytic, but not erythroid differentiation was induced. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that TIE was predominantly expressed in the human hematopoietic progenitor fraction, CD34+ cells. Fluorescence- activated cell sorting (FACS) showed that 42% of CD34+ and 17% of KIT- positive (KIT+) cells were TIE-positive (TIE+). The majority (81%) of the primitive hematopoietic stem cells, CD34+CD38- cells, were TIE+. Assays of progenitor cells and long-term culture-initiating cells (LTC- IC) showed that the TIE+ fraction contained more primitive cells than the TIE- fraction. Some TIE+ cells were in the CD34- fraction, which were CD19+ and CD20+ (B cells). These findings indicate that TIE has a unique spectrum of expression in primitive hematopoietic stem cells and B cells. Although its ligand has not been identified, TIE and its ligand may establish a novel regulatory pathway not only in early hematopoiesis, but also in the differentiation and/or proliferation of B cells.

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