Membrane-bound steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form

K. Miyazawa, D. A. Williams, A. Gotoh, J. Nishimaki, Hal Broxmeyer, K. Toyama

Research output: Contribution to journalArticle

211 Citations (Scopus)

Abstract

Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (Sl/Sl4) established from Sl/Sl homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, Sl4-h220 (membrane-bound form) induced more persistent activation of c-kit kinase than Sl4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that Sl4- h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas Sl4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to Sl4-h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a 'turn-off switch' for activated c- kit kinase.

Original languageEnglish (US)
Pages (from-to)641-649
Number of pages9
JournalBlood
Volume85
Issue number3
StatePublished - 1995
Externally publishedYes

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Stem Cell Factor
Protein-Tyrosine Kinases
Proteolysis
Protein Isoforms
Genes
Chemical activation
Proto-Oncogene Proteins c-kit
Membranes
Exons
Stromal Cells
Degradation
Proteins
Phosphotransferases
Complementary DNA
Cells
Cell Line
Kinetics
Alternative Splicing
Myeloid Cells
Methionine

ASJC Scopus subject areas

  • Hematology

Cite this

Membrane-bound steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form. / Miyazawa, K.; Williams, D. A.; Gotoh, A.; Nishimaki, J.; Broxmeyer, Hal; Toyama, K.

In: Blood, Vol. 85, No. 3, 1995, p. 641-649.

Research output: Contribution to journalArticle

Miyazawa, K. ; Williams, D. A. ; Gotoh, A. ; Nishimaki, J. ; Broxmeyer, Hal ; Toyama, K. / Membrane-bound steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form. In: Blood. 1995 ; Vol. 85, No. 3. pp. 641-649.
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abstract = "Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (Sl/Sl4) established from Sl/Sl homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, Sl4-h220 (membrane-bound form) induced more persistent activation of c-kit kinase than Sl4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that Sl4- h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas Sl4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to Sl4-h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a 'turn-off switch' for activated c- kit kinase.",
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