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 (SI/SI4) established from SI/SI 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, SI4-h220 (membrane-bound form) induced more persistent activation of c-kit kinase than SI4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that SI4-h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas SI4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to SI4-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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Alternative Splicing
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Amino Acid Sequence
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Animals
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Antibodies
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Cell Adhesion Molecules / metabolism
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Cell Line
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Cell Membrane / metabolism
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Electrophoresis, Polyacrylamide Gel
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Exons
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Gene Expression / drug effects
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Granulocyte-Macrophage Colony-Stimulating Factor / pharmacology
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Hematopoietic Cell Growth Factors / biosynthesis
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Hematopoietic Cell Growth Factors / metabolism*
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Hematopoietic Cell Growth Factors / pharmacology
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Humans
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Immunoblotting
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Interleukin-3 / pharmacology
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Kinetics
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Mice
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Molecular Sequence Data
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Peptides / immunology
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Phosphoproteins / isolation & purification
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Phosphoproteins / metabolism
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Phosphotyrosine
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Protein-Tyrosine Kinases / metabolism*
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Proto-Oncogene Proteins / biosynthesis*
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-kit
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Proto-Oncogenes*
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Receptor Protein-Tyrosine Kinases / biosynthesis*
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Receptor Protein-Tyrosine Kinases / genetics
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Receptor Protein-Tyrosine Kinases / metabolism
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Receptors, Colony-Stimulating Factor / biosynthesis*
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Receptors, Colony-Stimulating Factor / genetics
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Receptors, Colony-Stimulating Factor / metabolism
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / metabolism
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Recombinant Proteins / pharmacology
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Stem Cell Factor
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Transfection
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Tyrosine / analogs & derivatives
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Tyrosine / analysis
Substances
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Antibodies
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Cell Adhesion Molecules
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Hematopoietic Cell Growth Factors
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Interleukin-3
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Peptides
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Phosphoproteins
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Proto-Oncogene Proteins
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Receptors, Colony-Stimulating Factor
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Recombinant Proteins
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Stem Cell Factor
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Phosphotyrosine
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Tyrosine
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Granulocyte-Macrophage Colony-Stimulating Factor
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Protein-Tyrosine Kinases
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Proto-Oncogene Proteins c-kit
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Receptor Protein-Tyrosine Kinases