Comparative analysis of extra- and intracellular proteasomes from K562 cells

Abstract

In this manuscript we compared the biochemical properties of intracellular (cytoplasmic) and extracellular (secreted) proteasomes by their peptidase activities and the levels of phosphorylation of their subunits judged by relative mobility in two-dimensional gel electrophoresis and immunoblotting with phospho-specific antibodies. A comparative analysis of the peptidase activities of extra- and intracellular proteasomes showed that the excreted proteasomes exhibited higher chymotrypsin-like and lower trypsin-like peptidase activities than the cytoplasmic counterparts. The status of post-translational modifications, in particular phosphorylation, of 20S proteasomal subunits was also examined. We observed different levels of phosphorylation between the excreted and cytoplasmic proteasomes. According to the altered mobility in the two-dimensional electrophoresis of modified proteasome subunits combined with immunoblotting results using various phospho-specific antibodies, we concluded that subunits α2, α4, α7, and β7 were post-translationally modified. In addition, β2, β5, and α5 subunits, which possess enzymatic activities, were also differentially modified. Overall, the phosphorylation level of excreted proteasomes was lower than the intracellular ones. This observation indicates that the phosphorylation status of proteasomes may be important for their excretion from cells.