Purification of the colony-stimulating factor 1 receptor and demonstration of its tyrosine kinase activity.

Abstract

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. The CSF-1 receptor was purified from cell membranes of the J774.2 mouse macrophage cell line by solubilization with Triton X-100, CSF-1 affinity chromatography, and gel filtration. The purified receptor is a protein or glycoprotein of 165 kDa comprising a single polypeptide chain that is not covalently associated, either as a homopolymer, or with any other protein. CSF-1 stimulated autophosphorylation of the purified receptor in tyrosine residues. Casein but not histone was shown to act as a substrate for the tyrosine protein kinase activity of purified receptor. (3,7). Chemical crosslinking studies indicate that the cell surface CSF-1 receptor is a macromolecule of ==165,000 Mr that is not disulfide bonded to any other macromolecule in macrophage membranes (8). The CSF-1 receptor shares physical characteristics with, and is immunologically and functionally related to, the c-fms gene product (9, 10). In this paper, we describe the purification of the CSF-1 receptor and demonstrate that the purified receptor is a CSF-1-dependent tyrosine kinase. MATERIALS AND METHODSPurification of the CSF-1 Receptor. J774.2 mouse macrophage cells (11,12) were seeded (104 cells per ml) into 15 liters of prewarmed 10% (vol/vol) horse serum (Flow Laboratories) in alpha medium (Kansas City Biologicals, Lenexa, KS) and cultured at 37°C in a large spinner flask. After 4 days cells were harvested from log-phase cultures (=z5 x 105 cells per ml, >95% viable) and collected by centrifugation (500 x g for 10 min at 4°C); the cell pellet was resuspended in 5 volumes of ice-cold 4 mM iodoacetic acid in phosphate-buffered saline (0.14 mM NaCl/3 mM KCl/8 mM Na2HPO4/1.5 mM KH2PO4, pH 7.4). All the following procedures were done at 4°C.The methods for the preparation and solubilization of J774.2 cell membranes and the assay of the solubilized receptor have been described in detail (13). Briefly, the postnuclear supernatant fraction from Dounce-homogenized, hypotonically swollen cells was layered over 15% (wt/vol) sucrose and centrifuged at 100,000 x g for 30 min at 40C. The pellet was resuspended in 10-20 volumes of 10% (wt/vol) sucrose/50 mM NaCl/100 mM Tris HCl, pH 7.4, containing 10 ,g of leupeptin (Sigma) per ml, 1 unit of aprotinin (Sigma) per ml, and 1000 units of soybean trypsin inhibitor (type I-S, Sigma) per ml and adjusted to 1% (vol/vol) Triton X-100. After gentle Dounce homogenization with a loose-fitting piston the mixture was diluted with an equal volume of receptor-binding buffer (RBB)-10% (wt/vol) sucrose/50 mM NaCl/0.1% Triton X-100/3 mM NaN3/100 mM Tris HCl, pH 7.4-and centrifuged (100,000 x g for 45 min at 40C), and the supernatant was saved as the solubilized membrane fraction. This fraction (==12 ml) was further diluted with an equal volume of RBB and passed twice through the CSF-1-p-aminobenzamidoethyl (pABAE) Sepharose 4B (see below) column (1.2 x 3.5 cm) at a flow rate of 40-50 ml per hr. The ...

Interleukin 3 and phorbol ester stimulate tyrosine phosphorylation of overlapping substrate proteins

“…These studies have provided the conceptual framework to interpret results implicating kinase activity in the biochemical mechanisms of actions of the colony-stimulating factors. Each of the hemopoietic growth factors have now been molecularly cloned, but to date only the receptor for colony-stimulating factor 1 (CSF-1) has been characterized by sequence homology and enzyme activity to be a tyrosine kinase, homologous to the v-fins oncogene product [28].…”

Molecular Stress-inducing Compounds Increase Osteoclast Formation in a Heat Shock Factor 1 Protein-dependent Manner

“…Cells were then fixed and stained histochemically for TRAP and osteoclast numbers counted. For BMM preparation for Western blotting, bone marrow cells (10 6 cells/ml) were suspended in RPMI/HIFBS supplemented with 30% L-cell-conditioned medium (30,32), incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 3 days, and then, the non-adherent cell fraction was removed. The resulting adherent proliferating cells were then prepared for analysis.…”

CSF-1 Receptor Signaling in Myeloid Cells

“…This led to the identification (Guilbert and Stanley 1980) and purification (Yeung et al 1987) of the CSF-1 receptor (CSF-1R) and the demonstration that it possessed intrinsic tyrosine kinase activity (Yeung et al 1987). It was subsequently shown to be identical to the c-fms proto-oncoprotein previously studied by Sherr and colleagues .…”