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

Research output: Contribution to journalArticle

Abstract

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.

Details

Authors
External organisations
  • Ludwig Institute for Cancer Research, Uppsala branch
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • 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
Original languageEnglish
Pages (from-to)14192-14200
JournalJournal of Biological Chemistry
Volume270
Issue number23
Publication statusPublished - 1995
Publication categoryResearch
Peer-reviewedYes
Externally publishedYes

Bibliographic note

The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Experimental Clinical Chemistry (013016010)