PHOSPHORYLATION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR DURING INTERNALIZATION IN A-431 CELLS

To assess the functional activity of internalized epidermal growth fac tor (EGF) receptors in A-431 cells we investigated their ability to be both the target and activator of serine and threonine protein kinases . By incubating A-431 cells with EGF at 4 degrees C or at 37 degrees C , eluting surface-bound EGF with an acid buffer, and immunoprecipitati ng the EGF receptor with different antibodies, we were able to compare the phosphorylation state of internalized EGF receptors to those foun d on the plasma membrane in intact cells. Tryptic phosphopeptide mappi ng and subsequent phosphoamino acid analysis revealed four tyrosine, o ne threonine, and seven serine phosphorylation sites in the molecule o f plasma membrane receptor, while internalized EGF receptor contained one additional threonine and three additional serine phosphorylation s ites. Because acid-mediated removal of EGF from its receptor demonstra ted that the majority of EGF-induced phosphorylation required the cont inuous presence of activated receptor to be maintained, the conclusion was made that internalized EGF receptors may be not only the target o f protein kinases whose activity was detected in our assay but also an activator of at least some of them. The interaction between internali zed EGF receptors and nontyrosine protein kinase was also observed in vitro. Membrane-associated protein kinase was detected which phosphory lated a serine residue of the EGF receptor molecule in an EGF-dependen t manner. Subcellular fractionation revealed the presence of the serin e protein kinase both in the fractions of plasma membranes and high-de nsity endosomes. These results demonstrate that at its prelysosomal st age, internalization does not impair the functional activity of EGF re ceptor. (C) 1994 Academic Press, Inc.