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Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact cells

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Author

Summary, in English

The c-kit-encoded tyrosine kinase receptor for stem cell factor (Kit/SCFR) is crucial for the development of hematopoietic cells, melanoblasts, and germ cells. Ligand stimulation of Kit/SCFR leads to receptor dimerization and autophosphorylation on tyrosine residues. We recently showed, that protein kinase C (PKC) acts in an SCF-stimulated negative feedback loop, which controls Kit/SCFR tyrosine kinase activity and modulates the cellular responses to SCF (Blume-Jensen, P., Siegbahn, A., Stabel, S., Heldin, C.-H., and Ronnstrand, L. (1993) EMBO J. 12, 4199-4209). We present here the identification of the major phosphorylation sites for PKC in Kit/SCFR. Two serine residues in the kinase insert, Ser-741 and Ser-746, are PKC-dependent phosphorylation sites in vivo and account for all phosphorylation by PKC in vitro. Together they comprise more than 60% of the total SCF-stimulated receptor phosphorylation in living cells and 85-90% of its phosphorylation in resting cells. Two additional serine residues, Ser-821 close to the major tyrosine autophosphorylation site in the kinase domain and Ser-959 in the carboxyl terminus are SCF-stimulated PKC-dependent phosphorylation sites. However, they are not phosphorylated directly by PKC-alpha in vitro. Both specific receptor tyrosine autophosphorylation and specific receptor-associated phosphatidylinositide 3'-kinase activity was increased approximately 2-fold in response to SCF in PAE cells stably expressing Kit/SCFR(S741A/S746A). Furthermore, the kinase activity of Kit/SCFR(S741A/S746A) toward an exogenous substrate was increased, which was reflected as a decreased Km and an increased Vmax, in accordance with the negative regulatory role of PKC on Kit/SCFR signaling.

Publishing year

1995

Language

English

Pages

14192-14200

Publication/Series

Journal of Biological Chemistry

Volume

270

Issue

23

Document type

Journal article

Publisher

American Society for Biochemistry and Molecular Biology

Topic

  • Medicinal Chemistry

Keywords

  • Base SequenceHematopoietic Cell Growth Factors/pharmacologyMolecular Sequence Data*NaphthalenesPhosphatidylinositol 3-KinasesPhosphorylationPhosphotransferases (Alcohol Group Acceptor)/metabolismPolycyclic Compounds/pharmacologyProtein Kinase C/*physiologyProto-Oncogene Proteins/*metabolismProto-Oncogene Proteins c-kitReceptor Protein-Tyrosine Kinases/*metabolismReceptors
  • Colony-Stimulating Factor/*metabolismSignal TransductionStem Cell FactorTetradecanoylphorbol Acetate/pharmacologyTransfection

Status

Published

ISBN/ISSN/Other

  • ISSN: 1083-351X