Introduction: Evolving roles for ubiquitin in cellular regulation

CELLS RESPOND TO A COMPLEX array of internal and external signals, the consequences of which range from subtle alterations in the complement of catalytic, structural, or regulatory components in the short term to more profound long-term remodeling accompanying development and differentiation. The principal mechanism underlying this metabolic program has long been assumed to involve gene regulation; however, the last 10 years have witnessed a growing appreciation for the role of protein turnover as a fundamental mechanism for cellular control. This shift in thinking recognizes a basic principle of biochemistry: all metabolic processes exist as steady-state systems rather than as a simple two-state, “all-or-none” process. This concept is embodied in the dynamics between the simultaneous actions of protein kinases and phosphoprotein phosphatases in signal transduction cascades, the regulation of mRNA levels governing protein translation by altered rates of gene induction vs. mRNA stability, and the subsequent control of protein levels by differential changes in their rates of synthesis and degradation. The inherent inefficiency of this regulatory strategy is more than compensated for by the selective advantage provided by the speed with which systems can alter cellular responses by modulating the opposing steps without requiring de novo synthesis of additional machinery required to effect such changes. Although interest in the roles of protein turnover in cellular regulation has waxed and waned over the last several decades, the field enjoys a rich body of literature. In the late 1930s Schoenheimer (1) was the first to observe the dynamic balance between rates of synthesis and degradation governing cellular protein levels. Much later, Simpson (2) demonstrated that protein degradation in rat liver slices required metabolic energy and could be rapidly blocked by inhibitors of oxidative phosphorylation. Subsequent work from other groups has established a common requirement for ATP in the degradation of proteins within prokaryotes and eukaryotes. The paradox presented by an energy requirement for an inherently exothermic process was accentuated by the inability to demonstrate the effect in cell extracts, leading to speculation that the AlP requirement reflected a role in lysosomal acidification or membrane integrity. As late as 1976, in a landmark review by Goldberg and St. John (3), the mechanism (or mechanisms) underlying AlP-dependent protein degradation remained unresolved. Despite this major technical impasse, our understanding of the role for protein degradation in regulating cellular metabolism expanded rapidly in the 1960s and culminated in an encyclopedic review by Schimke and Doyle (4), who cataloged an impressive list of examples where the levels of key metabolic enzymes were controlled by differential regulation in their rates of protein synthesis and/or degradation. Regulatory proteins ranging from committed step enzymes within metabolic pathways to components of signal transduction cascades and factors required for gene transcription/repression generally possess short half-lives that are determined by structural features encoded within their amino acid sequences, but can be further modulated by ligand binding and post-translational modification. The half-life defines the rapidity with which protein levels adjust between different steady states in response to regulatory cues (5). The proportionality between half-life and the time required for a protein to shift steady-state levels is mathematically defined and holds both for proteins whose concentrations are declining as well as those whose concentrations are increasing due to gene induction (5). An equally important class of short-lived proteins are those having non-native2 structures due to misfolding, errors of cell trafficking, covalent modification, or environmental stress. With few exceptions, both classes of short-lived proteins are degraded by ATP-dependent mechanisms. Development of cell-free rabbit reticulocyte extracts exhibiting ATP-stimulated degradation of endogenous and model substrates provided the first breakthrough in elucidating the mechanism of energy-dependent proteolysis. Early work by Hershko, Rose, and co-workers (6-8) resolved this activity into two complementary fractions by anion exchange chromatography and identified the active component of the unadsorbed fraction as a low molecular weight, heat-stable protein termed ATP-dependent proteolysis factor 1 (APF-1),3 a name that anticipated a role for additional components. These studies reconcile the energy dependence of protein degradation as a requirement for ATP in the covalent conjugation of APF-1 to proteolytic substrates and were followed closely by the identification of APF-1 as the 8.6 kDa polypeptide ubiquitin, previously found linked by isopeptide bond to histone 2A in the high mobility group protein A24 (9). The ensuing rapid progress outlined a general mechanism for ubiquitin conjugation involving an ATP-dependent enzyme catalyzing the activation of the carboxyl terminus of ubiquitin (El), a Ii-