Macrophage colony-stimulating factor-induced tyrosine phosphorylation of c-fms proteins expressed in FDC-P1 and BALB/c 3T3 cells.
The c-fms protein is a receptor for macrophage colony-stimulating factor (M-CSF) with intrinsic protein-tyrosine kinase activity. We investigated the tyrosine phosphorylation of murine c-fms proteins expressed from a retroviral vector in factor-dependent myeloid FDC-P1 cells and in BALB/c 3T3 fibroblasts transformed by the expression of the c-fms gene. FDC-P1 cells expressing c-fms were able to grow and differentiate in response to M-CSF. Their c-fms proteins were normally phosphorylated on serine and became phosphorylated on tyrosine residues contained in five tryptic peptides when the cells were exposed to M-CSF. A subset of these peptides was constitutively phosphorylated in BALB/c cells expressing c-fms, consistent with the production of M-CSF by these cells. All the peptides detected in vivo were also phosphorylated in vitro. These peptides were analyzed by susceptibility to proteases, comparison with synthetic peptides, and site-directed mutagenesis. The identities of four of the tryptic peptides were determined; they arise from three unique tyrosine phosphorylation sites. One major site of tyrosine phosphorylation at residue 697 accounted for two of the tryptic peptides. A second major site was identified at tyrosine residue 706. These two tyrosine phosphorylation sites are located within the tyrosine kinase insert region. Tyrosine 807, which has homology to the major autophosphorylation site of the p60v-src tyrosine kinase, is a minor autophosphorylation site. Possible functional roles for these phosphorylations of the c-fms protein include interactions with substrate proteins, catalytic activity, and ligand-induced degradation.
Tapley
P
P
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Kazlauskas
A
A
Cooper
J A
JA
Rohrschneider
L R
LR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003115:Colony-Stimulating Factors / Q000494:pharmacology*; D016173:Macrophage Colony-Stimulating Factor; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; 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; D011994:Recombinant Proteins / Q000494:pharmacology; D014162:Transfection
D000596:Amino Acids; D003115:Colony-Stimulating Factors; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases; D016186:Receptor, Macrophage Colony-Stimulating Factor