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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

Blume-Jensen, Peter ; Wernstedt, Christer ; Heldin, Carl-Henrik and Rönnstrand, Lars LU (1995) In Journal of Biological Chemistry 270(23). p.14192-14200
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... (More)
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. (Less)
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published
subject
keywords
Base Sequence Hematopoietic Cell Growth Factors/pharmacology Molecular Sequence Data *Naphthalenes Phosphatidylinositol 3-Kinases Phosphorylation Phosphotransferases (Alcohol Group Acceptor)/metabolism Polycyclic Compounds/pharmacology Protein Kinase C/*physiology Proto-Oncogene Proteins/*metabolism Proto-Oncogene Proteins c-kit Receptor Protein-Tyrosine Kinases/*metabolism Receptors, Colony-Stimulating Factor/*metabolism Signal Transduction Stem Cell Factor Tetradecanoylphorbol Acetate/pharmacology Transfection
in
Journal of Biological Chemistry
volume
270
issue
23
pages
14192 - 14200
publisher
ASBMB
external identifiers
  • scopus:0029034468
ISSN
1083-351X
DOI
10.1074/jbc.270.23.14192
language
English
LU publication?
no
additional info
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)
id
d1c32b53-0d30-4afc-bb11-732e86bfdee9 (old id 1783980)
alternative location
http://www.jbc.org/content/270/23/14192.full
date added to LUP
2016-04-04 07:48:08
date last changed
2020-01-12 20:22:08
@article{d1c32b53-0d30-4afc-bb11-732e86bfdee9,
  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.},
  author       = {Blume-Jensen, Peter and Wernstedt, Christer and Heldin, Carl-Henrik and Rönnstrand, Lars},
  issn         = {1083-351X},
  language     = {eng},
  number       = {23},
  pages        = {14192--14200},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact cells},
  url          = {http://dx.doi.org/10.1074/jbc.270.23.14192},
  doi          = {10.1074/jbc.270.23.14192},
  volume       = {270},
  year         = {1995},
}