Identification of tyrosine 706 in the kinase insert as the major colony-stimulating factor 1 (CSF-1)-stimulated autophosphorylation site in the CSF-1 receptor in a murine macrophage cell line.
The receptor for colony-stimulating factor 1 (CSF-1) is a ligand-activated protein-tyrosine kinase. It has been shown previously that the CSF-1 receptor is phosphorylated on serine in vivo and that phosphorylation on tyrosine can be induced by stimulation with CSF-1. We studied the phosphorylation of the CSF-1 receptor by using the BAC1.2F5 murine macrophage cell line, which naturally expresses CSF-1 receptors. Two-dimensional tryptic phosphopeptide mapping showed that the CSF-1 receptor is phosphorylated on several different serine residues in vivo. Stimulation with CSF-1 at 37 degrees C resulted in rapid phosphorylation on tyrosine at one major site and one or two minor sites. We identified the major site as Tyr-706. The identity of Tyr-706 was confirmed by mutagenesis. This residue is located within the kinase insert domain. There was no evidence that Tyr-973 (equivalent to Tyr-969 in the human CSF-1 receptor) was phosphorylated following CSF-1 stimulation. When cells were stimulated with CSF-1 at 4 degrees C, additional phosphotyrosine-containing phosphopeptides were detected and the level of phosphorylation of the individual phosphotyrosine-containing phosphopeptides was substantially increased. In addition, we show that CSF-1 receptors are capable of autophosphorylation at six to eight major sites in vitro.
van der Geer
P
P
Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92138-9216.
Hunter
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003115:Colony-Stimulating Factors / Q000378:metabolism / Q000494:pharmacology*; D016173:Macrophage Colony-Stimulating Factor; D008264:Macrophages; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D009842:Oligopeptides / Q000138:chemical synthesis; D010447:Peptide Hydrolases; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016186:Receptor, Macrophage Colony-Stimulating Factor; D011956:Receptors, Cell Surface / Q000378:metabolism; D014443:Tyrosine
D003115:Colony-Stimulating Factors; D015345:Oligonucleotide Probes; D009842:Oligopeptides; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D014443:Tyrosine; D016173:Macrophage Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases; D016186:Receptor, Macrophage Colony-Stimulating Factor; D010447:Peptide Hydrolases