Kinetic Studies of the ADP-Ribosylation of HSP-83 in Chicken Embryo Fibroblast Cells

Abstract

ADP-ribosylation of proteins has been detected and characterized to occur as a posttranslational modification both on cytoplasmic and nuclear polypeptides. Usually, protein acceptors have been characterized by in vitro reactions, utilizing [³²P]-NAD⁺ with high specific activity as substrate for enzyme-catayzed transfer of ADP-ribose. Several cytoplasmic acceptors have in this way been identified, including such diverse proteins as the protein synthesis elongation factor 2 [1, 2] and several cytoplasmic structural proteins [3,4]. However, few studies have tried to answer the question of which proteins act as in vivo acceptors, mainly because of a lack of a suitable radio-actively labeled precursor. Recently I showed that the by far most abundant intracellular acceptor for mono(ADP-ribose) in vivo is a polypeptide with a M_r of 83,000 and identified by several criteria as identical with the stress-inducible and glucose-regulated HSP-83 [5]. This was made possible by utilizing [³H]-adenosine as a precursor to intracellular [³H]-ATP and thus also [³H]-NAD⁺, and separating the total cell homogenate by two-dimensional isoelectric focusing/SDS Polyacrylamide gel electrophoresis (2D IEF/SDS-PAGE) followed by fluorography. It was further shown that both heat shock and glucose starvation can induce drastic changes in the incorporation of tritiated ADP-ribose into HSP-83, suggesting that this modification of the protein plays an important physiological function. However, so far no enzymatic activity or other specific function has been assigned to HSP-83, other than its enhanced transcription during stress situations induced in a variety of ways [6].