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Cdc48-independent proteasomal degradation coincides with a reduced need for ubiquitylation.

ABSTRACT: Ubiquitin fusion degradation (UFD) substrates are delivered at the proteasome by a handover mechanism involving the ubiquitin-selective chaperone Cdc48 and the ubiquitin shuttle factor Rad23. Here, we show that introduction of a 20 amino acid peptide extension not only rendered degradation independent of Cdc48, in line with the model that this chaperone is involved in early unfolding events of tightly folded substrates, but at the same time relieved the need for efficient polyubiquitylation and the ubiquitin shuttle factor Rad23. Removal of the ubiquitylation sites in the N-terminal UFD signal made the degradation of this substrate strictly dependent on the peptide extension and also on Cdc48 and, importantly the presence of a functional ubiquitylation machinery. This suggests that the extension in the absence of N-terminal ubiquitylation sites is not properly positioned to engage the unfoldase machinery of the proteasome. Thus the need for efficient ubiquitylation and Cdc48 in facilitating proteasomal degradation are tightly linked but can be bypassed in the context of UFD substrates by the introduction of an unstructured extension. Our data suggest that polyubiquitin-binding complexes acting upstream of the proteasome, rather than the proteasome itself, can be primary determinants for the level of ubiquitylation required for protein degradation.

SUBMITTER: Godderz D

PROVIDER: S-EPMC5154593 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Cdc48-independent proteasomal degradation coincides with a reduced need for ubiquitylation.

Gödderz Daniela D Heinen Christian C Marchese Francesco P FP Kurz Tilman T Acs Klàra K Dantuma Nico P NP

Scientific reports 20150105

Ubiquitin fusion degradation (UFD) substrates are delivered at the proteasome by a handover mechanism involving the ubiquitin-selective chaperone Cdc48 and the ubiquitin shuttle factor Rad23. Here, we show that introduction of a 20 amino acid peptide extension not only rendered degradation independent of Cdc48, in line with the model that this chaperone is involved in early unfolding events of tightly folded substrates, but at the same time relieved the need for efficient polyubiquitylation and ...[more]

PMID: 25556859

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Project description:In chondrocytes, PTHrP maintains them in a proliferative state and prevents premature hypertrophy. The mechanism by which PTHrP does this is not fully understood. Both Runx2 and Runx3 are required for chondrocyte maturation. We recently demonstrated that cyclin D1 induces Runx2 protein phosphorylation and degradation. In the present studies, we tested the hypothesis that PTHrP regulates both Runx2 and Runx3 protein stability through cyclin D1. We analyzed the effects of cyclin D1 on Runx3 protein stability and function using COS cells, osteoprogenitor C3H10T1/2 cells and chondrogenic RCJ3.1C5.18 cells. We found that cyclin D1 induced Runx3 degradation in a dose-dependent manner and that both Myc-tagged Runx3 and endogenous Runx3 interact directly with CDK4 in COS and RCJ3.1C5.18 cells. A conserved CDK recognition site was identified in the C-terminal region of Runx3 by sequence analysis (residues 356-359). Pulse-chase experiments showed that the mutation of Runx3 at Ser356 to alanine (SA-Runx3) increased the half-life of Runx3. By contrast, the mutation at the same serine residue to glutamic acid (SE-Runx3) accelerated Runx3 degradation. In addition, SA-Runx3 was resistant to cyclin D1-induced degradation. GST-Runx3 was strongly phosphorylated by CDK4 in vitro. By contrast, CDK4 had no effect on the phosphorylation of SA-Runx3. Although both wild-type and SE-Runx3 were ubiquitylated, this was not the case for SA-Runx3. Runx3 degradation by cyclin D1 was completely blocked by the proteasome inhibitor PS1. In C3H10T1/2 cells, SA-Runx3 had a greater effect on reporter activity than SE-Runx3. The same was true for ALP activity in these cells. To investigate the role of cyclin D1 in chondrocyte proliferation and hypertrophy, we analyzed the growth plate morphology and expression of chondrocyte differentiation marker genes in Ccnd1-knockout mice. The proliferating and hypertrophic zones were significantly reduced and expression of chondrocyte differentiation marker genes and ALP activity were enhanced in 2-week-old Ccnd1-knockout mice. PTHrP significantly suppressed protein levels of both Runx2 and Runx3 in primary chondrocytes derived from wild-type mice. By contrast, the suppressive effect of PTHrP on Runx2 and Runx3 protein levels was completely abolished in primary chondrocytes derived from Ccnd1-knockout mice. Our findings demonstrate that the cell cycle proteins cyclin D1 and CDK4 induce Runx2 and Runx3 phosphorylation, ubiquitylation and proteasomal degradation. PTHrP suppresses Runx2 and Runx3 protein levels in chondrocytes through cyclin D1. These results suggest that PTHrP might prevent premature hypertrophy in chondrocytes, at least in part by inducing degradation of Runx2 and Runx3 in a cyclin-D1-dependent manner.

Dataset Information

Cdc48-independent proteasomal degradation coincides with a reduced need for ubiquitylation.

Publications

Cdc48-independent proteasomal degradation coincides with a reduced need for ubiquitylation.

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