Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.
Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.
Reith
A D
AD
Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Ellis
C
C
Lyman
S D
SD
Anderson
D M
DM
Williams
D E
DE
Bernstein
A
A
Pawson
T
T
D019870:1-Phosphatidylinositol 4-Kinase; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002461:Cell Line, Transformed; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D008407:Mast Cells; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D010957:Plasmids; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019009:Proto-Oncogene Proteins c-kit; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
D020690:GTPase-Activating Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D011505:Protein-Tyrosine Kinases; D019009:Proto-Oncogene Proteins c-kit; D010738:Type C Phospholipases