Activation of protein kinase C accelerates internalization of transferrin receptor but not of major histocompatibility complex class I, independent of their phosphorylation status.

Phosphorylation of membrane glycoproteins has often been invoked as a determinant of receptor internalization and receptor trafficking in a more general sense. Here we have studied the trafficking of major histocompatibility complex (MHC) Class I molecules and transferrin receptor (Tfr) related to their phosphorylation status in the human lymphoblastoid cell line JY. High resolution isoelectric focusing (IEF) allows the visualization of phosphorylated and non-phosphorylated protein species simultaneously, using protein backbone-labeling. Analysis on IEF was combined with a neuraminidase protection assay, in which sialic acid modification of the N-linked glycans present on Tfr and Class I molecules is used as a reporter group for cell surface expression. Phosphorylation of Class I heavy chains and Tfr was induced by exposure of cells to the phorbol ester tetradecanoyl phorbol acetate. We show that 1) phosphorylation of MHC Class I molecules is restricted to the cell surface fraction, 2) phosphorylation of MHC Class I molecules by protein kinase C (PKC) is not correlated with their internalization, as no internalization of Class I molecules, phosphorylated or non-phosphorylated, could be detected, 3) the initial rate, but not the final extent of the internalization of Tfr is affected by activation of PKC, and 4) phosphorylated Tfr behaves in a manner identical to non-phosphorylated Tfr in terms of internalization. The effect of activation of PKC on internalization of Tfr therefore most likely takes place at the level of the internalization machinery. Our data concerning the internalization of MHC Class I molecules contrast with earlier studies describing constitutive internalization in the B lymphoblastoid cell line A 46 and in HPB-ALL cells.