Differential effects of W mutations on p145c-kit tyrosine kinase activity and substrate interaction.
The c-kit gene, mapped to the dominant white spotting (W) locus of the mouse (Chabot, B., Stephenson, D. A., Chapman, V. M., Besmer, P., and Bernstein, A. (1988) Nature 335, 88-89; Geissler, E. N., Ryan, M. A., and Housman, D. E. (1988) Cell 55, 185-192), encodes a receptor tyrosine kinase, p145c-kit. Germline mutations at the W locus lead to loss of function alterations in p145c-kit, and result in mice with developmental defects of varying severity in the melanocytic, hematopoietic stem cell, and primordial germ cell lineages. To investigate in more detail the effect of W mutations on p145c-kit signaling, three mutations, W42, Wv, and W41, that confer severe, intermediate, and mild phenotypic characteristics, respectively, were introduced into the human p145c-kit tyrosine kinase domain. These mutations attenuated the intrinsic tyrosine kinase activity of the receptor to different degrees. In addition, they had differential effects on the interaction of the p145c-kit substrates, phospholipase C gamma, GTPase-activating protein, and the receptor-binding subunit of phosphatidylinositol 3'-kinase, p85. Notably, the Wv mutation, while retaining significant receptor tyrosine kinase activity, was unable to bind phospholipase C gamma and GTPase-activating protein, but could still associate with p85. These results suggest that the location of W mutations may be an important determinant of the specificity of substrate association and phosphorylation, and may explain, at least in part, the cell type-specific defects associated with certain W alleles.
Herbst
R
R
Department of Molecular Biology, Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Shearman
M S
MS
Obermeier
A
A
Schlessinger
J
J
Ullrich
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit; D013379:Substrate Specificity
D011518:Proto-Oncogene Proteins; D004815:Epidermal Growth Factor; D008715:Methionine; D011505:Protein-Tyrosine Kinases; D019009:Proto-Oncogene Proteins c-kit