ABSTRACT
The ubiquitin-dependent proteolysis system (UPS) is the main driver of regulated protein degradation in all eukaryotic cells, and it is becoming increasingly clear that defects within this pathway drive a large number of human pathologies. Recent success in the use of proteasome inhibitors in the treatment of hematological malignancies validates the UPS as a viable therapeutic pathway, and substantial effort is now focused on the development of both second-generation proteasome inhibitors as well as novel strategies for the inhibition of upstream UPS enzymes. In this review we discuss the potential 'druggability' of key nodes within the UPS and summarize recent advances within the field.
Subject(s)
Proteasome Inhibitors , Ubiquitin/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Humans , Neoplasms/drug therapy , Proteasome Endopeptidase Complex/metabolism , Protein Interaction Domains and Motifs/drug effects , Protein Processing, Post-Translational , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolismABSTRACT
To ensure proper timing of the G1-S transition in the cell cycle, the cyclin E-Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21(Cip1)/p27(Kip1)/p57(Kip2) family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27(Xic1) protein in Xenopus. Activation of cyclin E-Cdk2 is linked to the ubiquitination of human p27(Kip1) or Xenopus p27(Xic1) by SCF (for Skp1-Cullin-F-box protein) ubiquitin ligases. For human p27(Kip1), ubiquitination requires direct phosphorylation by cyclin E-Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E-Cdk2, but it does require nuclear accumulation of the Xic1-cyclin E-Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E-Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E-Cdk2 and the preinitiation complex. Thus, activation of the cyclin E-Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.
Subject(s)
CDC2-CDC28 Kinases , Calcium-Binding Proteins , Cell Cycle Proteins/metabolism , Cell Cycle/physiology , Cullin Proteins , Cyclin E/metabolism , Cyclin-Dependent Kinases/metabolism , DNA Replication/physiology , Nuclear Proteins , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins , Ubiquitins/metabolism , Xenopus Proteins , Animals , Carrier Proteins , Cell Cycle Proteins/genetics , Chromatin/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Cyclin E/genetics , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Female , Ligases/genetics , Ligases/metabolism , Oocytes/cytology , Oocytes/metabolism , Origin Recognition Complex , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Peptide Synthases/genetics , Peptide Synthases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/genetics , S-Phase Kinase-Associated Proteins , SKP Cullin F-Box Protein Ligases , Ubiquitin-Protein Ligases , Ubiquitins/genetics , Xenopus laevisABSTRACT
Recently, many new examples of E3 ubiquitin ligases or E3 enzymes have been found to regulate a host of cellular processes. These E3 enzymes direct the formation of multiubiquitin chains on specific protein substrates, and - typically - the subsequent destruction of those proteins. We discuss how the modular architecture of E3 enzymes connects one of two distinct classes of catalytic domains to a wide range of substrate-binding domains. In one catalytic class, a HECT domain transfers ubiquitin directly to substrate bound to a non-catalytic domain. Members of the other catalytic class, found in the SCF, VBC and APC complexes, use a RING finger domain to facilitate ubiquitylation. The separable substrate-recognition domains of E3 enzymes provides a flexible means of linking a conserved ubiquitylation function to potentially thousands of ubiquitylated substrates in eukaryotic cells.
Subject(s)
Ligases/metabolism , Animals , Catalytic Domain , Eukaryotic Cells/enzymology , Humans , Ligases/chemistry , Substrate Specificity/physiology , Ubiquitin-Protein LigasesABSTRACT
The SRm160/300 splicing coactivator, which consists of the serine/arginine (SR)-related nuclear matrix protein of 160 kDa and a 300-kDa nuclear matrix antigen, functions in splicing by promoting critical interactions between splicing factors bound to pre-mRNA, including snRNPs and SR family proteins. In this article we report the isolation of a cDNA encoding the 300-kDa antigen and investigate the activity of it and SRm160 in splicing. Like SRm160, the 300-kDa antigen contains domains rich in alternating S and R residues but lacks an RNA recognition motif; the protein is accordingly named "SRm300." SRm300 also contains a novel and highly conserved N-terminal domain, several unique repeated motifs rich in S, R, and proline residues, and two very long polyserine tracts. Surprisingly, specific depletion of SRm300 does not prevent the splicing of pre-mRNAs shown previously to require SRm160/300. Addition of recombinant SRm160 alone to SRm160/300-depleted reactions specifically activates splicing. The results indicate that SRm160 may be the more critical component of the SRm160/300 coactivator in the splicing of SRm160/300-dependent pre-mRNAs.
Subject(s)
Antigens, Nuclear , Nuclear Matrix-Associated Proteins , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA Splicing/genetics , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Antibodies, Monoclonal , Cell Nucleus/metabolism , Cells, Cultured , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , DNA, Complementary/metabolism , Humans , Molecular Sequence Data , Nuclear Proteins/immunology , Precipitin Tests , RNA Splicing/physiology , RNA, Messenger/metabolism , Spliceosomes/metabolismABSTRACT
Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. We have recently identified a splicing "coactivator," SRm160/300, which contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. In the present study, we show that SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independently of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound "activators" and the snRNP machinery of the spliceosome.
