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The serine/threonine phosphatase PP5 interacts with CDC16 and CDC27, two tetratricopeptide repeat-containing subunits
1Department of Chemistry and Biochemistry and the Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0367, USA.
Abstract:
The evolutionarily conserved multisubunit complex known as the cyclosome or anaphase-promoting complex is involved in catalyzing the ubiquitination of diverse substrates in M phase, allowing their destruction by the 26 S proteasome and the completion of mitosis. Three of the eight subunits of the anaphase-promoting complex (CDC16, CDC23, and CDC27) have been shown to be phosphorylated in M phase, and their phosphorylation is required for the anaphase-promoting complex to be active as a ubiquitin ligase. Several subunits of the anaphase-promoting complex contain tetratricopeptide repeats, a protein motif involved in protein/protein interactions. PP5 is a serine/threonine phosphatase that also contains four copies of the tetratricopeptide repeats motif. Here we show by a combination of two-hybrid analysis and in vitro binding that PP5 interacts with CDC16 and CDC27, two subunits of the anaphase-promoting complex. Only the NH2-terminal domain of PP5, containing all four tetratricopeptide repeats, is required for this physical interaction. Deletion analysis suggests that the site of binding to PP5 is localized to the COOH-terminal block of tetratricopeptide repeats in CDC16 and CDC27. In addition, indirect immunofluorescence showed that PP5 localizes to the mitotic spindle apparatus. The direct interaction of PP5 with CDC16 and CDC27, as well as its overlapping spindle localization in mitosis, suggests that PP5 may be involved in the regulation of the activity of the anaphase-promoting complex.
Insights
The phosphatase PP5 interacts with CDC16 and CDC27 subunits of the anaphase-promoting complex (APC/C). This interaction, localized to the mitotic spindle, suggests PP5 regulates APC/C activity during mitosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The anaphase-promoting complex (APC/C) is a crucial E3 ubiquitin ligase regulating mitosis.
- APC/C activity depends on phosphorylation of its subunits, including CDC16, CDC23, and CDC27.
- Tetratricopeptide repeat (TPR) motifs mediate protein-protein interactions.
Purpose of the Study:
- To investigate the interaction between PP5 and APC/C subunits.
- To identify the domains involved in the PP5-APC/C interaction.
- To determine the cellular localization of PP5 during mitosis.
Main Methods:
- Two-hybrid analysis to detect protein-protein interactions.
- In vitro binding assays to confirm interactions.
- Deletion analysis to map interaction domains.
- Indirect immunofluorescence microscopy to determine cellular localization.
Main Results:
- PP5 directly interacts with APC/C subunits CDC16 and CDC27.
- The N-terminal TPR domain of PP5 mediates this interaction.
- Binding sites on CDC16 and CDC27 are located within their C-terminal TPR domains.
- PP5 localizes to the mitotic spindle apparatus.
Conclusions:
- PP5 physically interacts with key APC/C subunits.
- PP5's localization overlaps with APC/C on the mitotic spindle.
- These findings suggest PP5 plays a regulatory role in APC/C activity during mitosis.