Synthesis and Phosphorylation of Plasma Membrane Proteins of Friend Erythroleukemia Cells Induced to Differentiate

Friend virus-transformed murine erythroleukemia (FL) cells, either untreated or induced to differentiate by treatment with hexamethylene bisacetamide, were fractionated and subjected to polyacrylamide gel electrophoresis. Analysis of the fractions of the treated cells revealed no changes in either the rate of synthesis or the accumulation of the membrane proteins, including protein bands comigrating with spectrin, actin, and Band 3, as compared to the fractions of the untreated cells. Only a small fraction of a protein band comigrating with spectrin was associated with the plasma membrane. The bulk of the spectrin was detected in the cytosol cross-linked via S—S bonds to form aggregates. Protein bands comigrating with actin and Band 3 were distributed equally between the plasma membrane and the cytosol fractions. However, differences between untreated and induced cells were observed in regard to the pattern of phosphorylation of the proteins. Dephosphorylation of the proteins of the differentiating cells, particularly those of the plasma membrane, could be detected shortly after treatment. Turnover of labeled phosphate in spectrin molecules from hexamethylene bisace-tamide-induced, as well as uninduced, cells was very slow. Although actin from uninduced cells incorporated labeled phosphate very rapidly, in treated cells, the rate of incorporation decreased 24 hr after onset of differentiation. In addition, the plasma membrane proteins differed from those obtained from normal erythroid cells in several respects. (a) Turnover of phosphate in spectrin was more rapid in reticulocytes and mature erythrocytes as compared to the amount of label detected in the comigrating band of spectrin from induced or uninduced FL cells. (b) FL cell spectrin bands migrate with an apparent molecular weight of about 200, 000 to 220, 000, whereas normal erythrocyte spectrin bands migrate with a molecular weight of 200, 000 to 240, 000. And (c) Band 3, M.W. 100, 000 protein, although comigrating with the mouse erythrocyte Band 3, was heterologous rather than homologous. These data suggest that the plasma membrane proteins of FL cells may be of an abnormal type.