Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact cells.
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 Rönnstrand, 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.
Blume-Jensen
P
P
Ludwig Institute for Cancer Research, Uppsala Branch, Biomedical Center, Sweden.
Wernstedt
C
C
Heldin
C H
CH
Rönnstrand
L
L
D001483:Base Sequence; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D008969:Molecular Sequence Data; D009281:Naphthalenes; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D016184:Receptors, Colony-Stimulating Factor / Q000378:metabolism*; D015398:Signal Transduction; D019089:Stem Cell Factor; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D016298:Hematopoietic Cell Growth Factors; D009281:Naphthalenes; D011083:Polycyclic Compounds; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D019089:Stem Cell Factor; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases; D011493:Protein Kinase C; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate