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 homezygous murine embryo fetal liver and its stable transfectants containing either hSCF(Z48) cDNA (including exon 6; secreted form) or hSCF(220) 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-kif 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 [S-35] 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-kif 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-hit 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. (C) 1995 by The American Society of Hematology.