Purification and characterization of the erythropoietin-sensitive membrane phosphoprotein, pp43.
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.
Choi
H S
HS
Division of Hematology and Oncology, New England Deaconess Hospital, Boston, Massachusetts.
Bailey
S C
SC
Donahue
K A
KA
Vanasse
G J
GJ
Sytkowski
A J
AJ
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D004921:Erythropoietin / Q000494:pharmacology*; D007118:Immunoassay; D004915:Leukemia, Erythroblastic, Acute; D007942:Leukemia, Experimental; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation
D000939:Epitopes; D008565:Membrane Proteins; D010750:Phosphoproteins; D004921:Erythropoietin