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Association with cullin partners protects ROC proteins from proteasome-dependent degradation
1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599-7295, USA.
Abstract:
Cullin 1/CDC53 represents a multigene family and has been linked to the ubiquitin-mediated proteolysis of several different proteins. We recently identified two closely related RING finger proteins, ROC1 and ROC2, that share considerable sequence similarity to an APC subunit, APC11, and demonstrated ROC1 as an essential subunit of CUL1 and CDC53 ubiquitin ligases. We report here that the expression of ROC1, ROC2 and APC11 genes are induced by mitogens and remain constant during the cell cycle. Unlike other subunits of SCF and APC E3 ligases, ectopically expressed ROC family proteins are degraded by a proteasome-inhibitor sensitive pathway and are stabilized by associating with cullins. Mutations at the conserved Phe79 and His80 residues in the RING finger of ROC1 diminish its binding with cullins, resulting in a loss of cullin protection and ubiquitin ligase activity. These results suggest a potential mechanism for regulating the activity of ROC-cullin ligases through complex assembly and ROC/APC11 subunit ubiquitination.
Insights
ROC1 and ROC2 proteins are essential for ubiquitin ligase activity. Their stability and function depend on association with cullins, suggesting a novel regulatory mechanism for E3 ligases.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Cullin 1/CDC53 proteins are involved in ubiquitin-mediated proteolysis.
- ROC1 and ROC2 are related RING finger proteins similar to APC11.
- ROC1 is a crucial component of CUL1 and CDC53 ubiquitin ligases.
Purpose of the Study:
- To investigate the regulation and function of ROC1, ROC2, and APC11.
- To understand the role of cullin association in ROC protein stability and ligase activity.
Main Methods:
- Gene expression analysis of ROC1, ROC2, and APC11.
- Analysis of ectopically expressed ROC proteins stability.
- Site-directed mutagenesis of ROC1 RING finger residues.
- Assessment of ubiquitin ligase activity.
Main Results:
- ROC1, ROC2, and APC11 gene expression is induced by mitogens and cell cycle-constant.
- Ectopically expressed ROC proteins are proteasome-degraded but stabilized by cullin association.
- Mutations in ROC1's RING finger impair cullin binding, stability, and ligase activity.
Conclusions:
- ROC-cullin ligase activity is regulated by complex assembly and ROC/APC11 ubiquitination.
- Cullin association is critical for protecting ROC proteins from degradation and maintaining ligase function.