Membrane-bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form.
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.
Miyazawa
K
K
First Department of Internal Medicine (Hematology and Oncology), Tokyo Medical College, Japan.
Williams
D A
DA
Gotoh
A
A
Nishimaki
J
J
Broxmeyer
H E
HE
Toyama
K
K
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D015870:Gene Expression / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D016298:Hematopoietic Cell Growth Factors / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007377:Interleukin-3 / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit; D011519:Proto-Oncogenes; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D016184:Receptors, Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D019089:Stem Cell Factor; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D000906:Antibodies; D015815:Cell Adhesion Molecules; D016298:Hematopoietic Cell Growth Factors; D007377:Interleukin-3; D010455:Peptides; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D019000:Phosphotyrosine; D014443:Tyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases