Abstract
A 32P-labeled protein that co-purified with acidic fibroblast growth factor (aFGF) receptor from bovine liver proved to be a distinct membrane protein, which itself has kinase activity that is stimulated by aFGF. The protein was designated MAFP for major aFGF-stimulated phosphoprotein. MAFP was purified from bovine liver using immunoaffinity chromatography with monoclonal antibody to MAFP following Triton X-100 extraction of plasma membranes and wheat germ lectin-Sepharose 4B column chromatography. The purified MAFP showed molecular masses of 130 kDa and 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. Purified MAFP elicited aFGF-stimulated Thr-specific autophosphorylation activity and phosphorylation activity toward protein substrates (myelin basic protein and histone). Amino acid sequence analyses of 16 peptide fragments of MAFP, produced by endoproteinase Lys-C digestion followed by reduction and S-pyridylethylation, showed approximately 80-100% homology with the cDNA-deduced amino acid sequences of human and mouse plasma cell membrane glycoprotein, PC-1 (Buckley, M. F., Loveland, K. A., McKinstry, W. J., Garson, O. M., and Goding, J. W. (1990) J. Biol. Chem. 265, 17506-17511), suggesting that MAFP is the bovine version of PC-1. The amino acid sequences of bovine MAFP, human and mouse PC-1 reveal a putative ATP binding site in their extracellular domains. These results suggest that MAFP(PC-1) is an ectoprotein kinase. In addition to the kinase activity, MAFP(PC-1) was also found to possess alkaline nucleotide phosphodiesterase activity. It is now clear that several of the unique properties previously attributed to the aFGF receptor kinase are actually properties of this novel Thr-specific ectoprotein kinase, which co-purifies with the aFGF receptor and is responsive to stimulation by aFGF.
Highlights
AS2P-labeled protein that co-purified with acidic ent on cells of lymph nodes, hemolytic plaque-forming cells fibroblast growth factor receptor from bovine of spleen and myelomas, and cells of non-lymphoid tissues
Preparation of Partially PurifiedaFGF Receptor-The acidic fibroblast growth factor (aFGF) recep- receptor was partially purified from bovine liverby Triton X
Clonal antibodies raised to this preparationled to the discovappears to co-purify with the aFGF receptor during purification of ery that MAFPis a distinct protein that co-chromatographed the latter (6),partially purified aFGF receptor from DEAE-cellulose columnchromatography,asdescribed above,wasused as antigen
Summary
AS2P-labeled protein that co-purified with acidic ent on cells of lymph nodes, hemolytic plaque-forming cells fibroblast growth factor (aFGF) receptor from bovine of spleen and myelomas, and cells of non-lymphoid tissues. The deduced amino acid sequence of bovine liverusingimmunoaffinitychromatography with monoclonal antibody to MAFP following Triton X-100 extraction of plasmamembranesandwheat germlectin-Sepharose 4B column chromatography.
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