The Ubiquitin-Proteasome Proteolytic System

The following sections are included:

• Preface

• Contents

The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin-mediated proteolytic system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. The ligation of ubiquitin to protein involves the successive action of three types of enzymes: the ubiquitin-activating enzyme E1, a ubiquitin-carrier protein E2 and a ubiquitin-protein ligase, E3. The selectivity and the regulation of the degradation of a specific protein are usually determined by the properties of its specific ubiquitin ligase (E3) enzyme. Recently we have been studying two ubiquitin ligase complexes that have important roles in different aspects of cell cycle regulation. One is the cyclosome, or Anaphase-Promoting Complex (APC), which acts on mitotic cyclins and some other regulators in exit from mitosis. The cyclosome is activated at the end of mitosis by phosphorylation, a process that allows its further activation by the ancillary protein Cdc20. A different complex, which belongs to the SCF (Skp1-Cullin-F-box protein) family of ubiquitin ligases, is involved in the degradation of p27, a mammalian G1 cyclin-dependent kinase (Cdk) inhibitor, following mitogenic stimulation. Its action is triggered by Cdk2-dependent phosphorylation of p27, as well as by the increase in levels of a specific F-box protein Skp2 and of the cell cycle regulatory protein Cks1, that all take place in the G1 to S-phase transition…

Eukaryotes contain a highly conserved multienzyme system which covalently links a small protein ubiquitin (Ub) to a variety of intracellular proteins that bear degradation signals recognized by this system. The resulting Ub-protein conjugates are degraded by the 26S proteasome, an ATP-dependent multisubunit protease. Pathways that involve Ub play major roles in a huge variety of processes, including cell differentiation, cell cycle, and responses to stress. In this paper, I briefly review the design of the Ub system, describe in more detail one of its pathways, called the N-end rule pathway, and consider three recent discoveries: the finding that ubiquitin ligases interact with specific components of the 26S proteasome (1), the finding that peptides accelerate their uptake into cells by activating the N-end rule pathway (2), and the finding that the degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability (3).

The selection of protein substrates for ubiquitination is commonly achieved by the specific interactions of E3 protein-ubiquitin ligases with both target proteins and E2 enzymes. The protein-protein interactions that confer selectivity to the ubiquitination machinery have many features in common with the modular protein interactions characteristic of signal transduction pathways. Notably, recruitment of a target protein into an E3 protein-ubiquitin ligase complex is often dependent on its phosphorylation on either serine/threonine or tyrosine residues. We discuss examples of phospho-dependent protein ubiquitination in signal transduction and the cell cycle, and suggest that viral proteins, such as the Epstein-Barr virus polypeptide LMP2A, can stimulate the ubiquitination of host cell proteins by serving as a scaffold to recruit protein-ubiquitin ligases and their substrates. We summarize recent data suggesting that multi-site phosphorylation of the yeast CDK inhibitor Sic1 is required for its binding to an E3 complex, and consequent destruction. Based on experimental observation and theoretical modeling, we argue that this requirement for multi-site phosphorylation of Sic1 during the G1 phase of the cell cycle yields a switch-like effect which is important for regulating entry into S phase.

The following sections are included:

• Introduction

• The 20S Proteasome

• Archaeal and Bacterial Activators of the 20S Proteasome

• The 19S Regulatory Complex

• Structural Features of the 26S Proteasome

• Conclusions

• References

In most cases, target substrates of the ubiquitin proteolytic system are completely degraded. In several exceptions, such as the first step in the activation of the transcriptional regulator NF-κB, the substrate-the precursor protein p105—is processed in a limited manner to yield p50 (residues 1-435), an active subunit of the heterodimeric factor. p50 is derived from the N-terminal domain of p105, whereas the C-terminal, ankyrin repeat-containing domain, is degraded. The mechanisms involved in this unique process have remained largely obscure. A Gly-rich region (GRR) in the C-terminal domain of p50 acts as a "stop" signal and interferes with processing of the ubiquitinated precursor by the 26S proteasome. In addition, p105 contains two distinct targeting motifs that act under different physiological conditions. In the resting cell, Lys residues 441 and 442 serve as ubiquitination targets, whereas the neighboring downstream acidic sequence (residues 446-454), can function as a ligase recognition motif. Following IκB kinase (IKK)-mediated phosphorylation of Ser residues in the C-terminal domain of p105 (amino acids 918-934), the SCF^β-TrCP ubiquitin ligase is recruited, and ubiquitination of yet unknown Lys residues leads to accelerated processing. Ubiquitin conjugation and subsequent processing of a series of precursors of p105 that lack the C-terminal signaling domain, but still contain the ankyrin repeat domain, is progressively inhibited with increasing number of repeats. Inhibition is due to docking of active NF-κB subunits to the repeats. Inhibition is alleviated by phosphorylation and ubiquitination of the C-terminal signaling domain that result in degradation of the ankyrin repeat domain and release of the anchored subunits. A model is proposed that may explain the requirement for two sites for p105 processing. Under basal conditions, p50 is generated cotranslationally or via slow processing of mature p105 that involves the mid-molecule site. Following signaling, the C-terminal site is involved in rapid processing/degradation of the mature molecule with release of a large amount of stored, transcriptionally active subunits.

Growth factors and their transmembrane receptor tyrosine kinases play essential roles in survival, proliferation and migration of both normal and tumor cells. An example is provided by the ErbB/HER family of receptors and the multiple neuregulin and EGF-like ligands. The major route leading to negative regulation of signaling downstream of these receptors involves endocytic removal of ligand-receptor complexes from the cell surface. Ubiquitination of receptor tyrosine kinases such as ErbBs plays a central role in their down-regulation, therefore research into the mechanism and role of receptor ubiquitination is tightly coupled to that of receptor endocytosis. Apparently several distinct endocytic itineraries exist: A constitutive pathway acts to maintain a steady state of signaling-competent receptors. In contrast, ligand stimulation and kinase activation triggers a rapid course of receptor endocytosis, resulting in signal termination. This pathway favors ErbB-1 over the other ErbB-family members. A third pathway involving chaperones and co-chaperones displays preference for ErbB-2, the most oncogenic member of the ErbB/HER family. Ubiquitination at or close to the plasma membrane may be a common feature of all three pathways. However, the exact endocytic route and the final degradative destination, namely the lysosome or the proteasome, appear to significantly differ. Additionally, these pathways are distinguishable in terms of the sorting mechanisms, the effect of kinase activity, and the involvement of specific ubiquitin ligases. Molecular mechanisms underlying degradation of growth factor receptors by both the ligand-induced pathway and the by chaperone-mediated route are discussed and open questions are identified. Detailed understanding of the complex machineries regulating endocytic inactivation of growth factor receptors holds promise for cancer therapy: antibody-induced endocytosis of ErbB-1 and ErbB-2 effectively blocks some types of human cancer, and low molecular weight drugs that target ErbB receptors to destruction through the stress-related pathway are currently being tested in patients.

The following sections are included:

• Cell Cycle Regulation by Cyclin-Dependent Kinases

• Features and Functions of p27

• Regulation of p27 Protein Levels by the Ubiquitin-Proteasome Pathway

• Regulation of the G1/S Transition by SCF Complexes

• SCF^Skp2

• Cks1

• Skp2 and Oncogenesis

• Conclusions

• Acknowledgments

• References

The following sections are included:

• The Cytokine/Hematopoietin Receptor Superfamily

• Signaling Mechanisms of the GHR

• Negative Regulators of GH Signaling

• Ubiquitination of Plasma Membrane Proteins

• The Ubiquitin System and the Stress Response

• References

The following sections are included:

• Introduction

• The Art of Hiding

• The GAr is a Transferable Modular Signal

• Repeat Composition and Length

• Mode of Action

• Other Stabilization Signals

• Concluding Remarks

• Acknowledgments

• References

Autosomal recessive juvenile parkinsonism (AR-JP) is the most prevalent form of familial Parkinson's disease (PD). AR-JP is characterized by selective and exhaustive loss of dopaminergic neurons in the substantia nigra of the midbrain. Clinical features of AR-JP resemble those of idiopathic PD, but AR-JP lacks Lewy bodies, the pathological hallmark of idiopathic PD. Parkin, the causative gene of AR-JP, encodes a 52-kDa protein that is a RING-finger type ubiquitin (Ub)-protein ligase (E3) collaborating with an Ub-conjugating enzyme (E2) belonging to a cognate class of UbcH7 or UbcH8. Analysis of parkin mutations in AP-JP patients reveals that the functional loss of parkin as an E3 enzyme is the molecular basis of AR-JP, suggesting that accumulation of as-yet-unidentified protein(s) causes nigral neuron death. Thus, it is clear that abnormalities of proteolysis mediated by the Ub-proteasome pathway are involved in AR-JP pathogenesis. These findings should shed new light on the mechanisms underlying neurodegeneration in idiopathic PD as well as AR-JP.