Purification and characterization of the erythropoietin-sensitive membrane phosphoprotein, pp43

• Published in: The Journal of biological chemistry Vol. 265; no. 7; pp. 4143 - 4148
• Main Authors: Choi, H S, Bailey, S C, Donahue, K A, Vanasse, G J, Sytkowski, A J
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05-03-1990
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cell Fractionation; Cell Line; Cell Membrane - metabolism; cell membranes; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Epitopes - analysis; erythroid cells; erythropoietin; Erythropoietin - pharmacology; Fundamental and applied biological sciences. Psychology; Immunoassay; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Membrane Proteins - immunology; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Mice; Miscellaneous; Molecular Weight; Phosphoproteins - immunology; Phosphoproteins - isolation & purification; Phosphoproteins - metabolism; Phosphorylation; Proteins; Hormone; Phosphorylation; Purification; Sensitivity resistance; Erythropoietin; Phosphoproteins; Plasma membrane; Glycoproteins; Localization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)39714-5

We have shown previously that purified human erythropoietin rapidly alters the phosphorylation of an integral erythroid membrane
protein, pp43 (Choi, H.-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933-2936). We have now purified
pp43 to apparent homogeneity and have prepared antibodies to it. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and electrophoretic transfer of membrane proteins to nitrocellulose, the antibodies identified pp43 and a series of higher
molecular weight antigenically related proteins, up to 50 kDa, in erythropoietin-responsive Rauscher murine erythroleukemia
cells and in normal murine erythroid cells. Examination of purified subcellular fractions confirmed the localization of pp43
and the related proteins to the plasma membrane. Phosphorylation with [gamma-32P]ATP demonstrated that, in contrast to pp43,
these higher molecular weight proteins were not phosphorylated. Marked differences in both the abundance of pp43 and related
proteins and the degree of erythropoietin-sensitive pp43 phosphorylation were found between the plasma membranes of Rauscher
cells and those of "non-responsive" Friend murine erythroleukemia cells. In addition only trace amounts of a 50-kDa antigenically
related protein and no phosphorylated pp43 were detected in the plasma membranes of two erythropoietin-insensitive human erythroid
cells lines, K562 and HEL. The results suggest that the abundance and degree of phosphorylation of pp43 and the antigenically
related proteins is strongly correlated with the erythropoietin responsiveness of the particular erythroid cell types.