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.

Abstract

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. Twodimensional tryptic phosphopeptide mapping showed that the CSF-1 receptor is phosphorylated on several different serine residues in vivo. Stimulation with CSF-1 at 3rC 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°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.

The kinase insert domain of colony stimulating factor‐1 receptor is dispensable for CSF‐1 induced phosphatidylcholine hydrolysis

“…Mouse and human CSF1R mutants devoid ¢3f the KI domain do not associate well with Ptdlns-3 kinase and are mitogenicaIIy impaired in transfected fibroblasts [3,4,25]. Furthermore, two major tyrosine autophosphorylation sites have been mapped in the KI domain of the mouse receptor (the positions of the homologous residues in human CSFIR are Y699 and Y708) [26,27] (Fig.3A). We e×pressed a 67 amino acid KI domain deletion mutant (Gly-684), spanning from glycine 684 to leucine 750, in NIH 3T3 cells [3].…”

Deletion or substitution within the alpha platelet-derived growth factor receptor kinase insert domain: effects on functional coupling with intracellular signaling pathways.

“…Receptor autophosphorylation, an indirect measure of tyrosine kinase activity, was progressively impaired by the deletion mutants. The cxRAki-1 mutant lacks tyrosine residues, which have been shown to be sites of tyrosine phosphorylation in PPDGF and CSF-1 receptors (25,44,48). Therefore, the 10-fold reduction in receptor phosphorylation of the aRAki-1 mutant may be due in part to the loss of this putative autophosphorylation site.…”

“…Earlier studies have shown that PDGF induces phosphorylation of its own receptor on tyrosine residues (12). Sites of receptor autophosphorylation have been mapped within the ki domains of both PDGFR (25) and CSF-1R (44,48). The aLPDGFR ki domain contains analogous tyrosine residues, which are deleted in both aPDGFR ki mutants.…”

“…However, this enzyme has been shown to be coimmunoprecipitated with activated PPDGFR (7) CSF-1R (49). Moreover, mutation or loss of autophosphorylation sites located within the respective ki domains of the ,PDGFR (25) and CSF-1R (42,48) has been shown to diminish the ability of these mutant receptors to associate with PI-3 kinase. aPDGFR-associated PI-3 kinase activity was readily detectable and comparable in PDGF-BB-stimulated 32D-aR and aRAki-fms cells (Fig.…”

Proteomic Analysis of Macrophage Differentiation

“…CSF-1 binds to the class IV cytokine receptor family member, the CSF-1R, which is the product of the c-fms proto-oncogene (2). Upon ligand binding the CSF-1R undergoes dimerization and subsequent auto-and trans-phosphorylation (3)(4)(5). The phosphorylation of the CSF-1R is thought to create docking sites for downstream signal transduction molecules containing SH2 domains (6 -8).…”