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.
Highlights
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
Immunofluorescence studies in HeLa cells have shown that a portion of the cellular pool of CDC16 and CDC27 localizes to the spindle/centrosome throughout M phase, where it most likely acts as part of anaphase-promoting complex (APC) to catalyze the ubiquitination of diverse factors (9)
In an effort to determine whether PP5 may be involved in regulating the activity of APC, we have examined its physical interaction with two subunits of APC (CDC16 and CDC27) which are phosphorylated during M phase
Summary
Constructs—All constructs were made using standard techniques. Each construct was sequenced to verify the correct frame as well as the proper sequence of any linker introduced during the cloning procedure. Xenopus and Mouse PP5 cDNAs—A full-length mouse PP5 cDNA was isolated from a macrophage -Zap library (kindly provided by Dr Chris Glass, University of California, San Diego), using a probe encoding part of the NH2-terminal TPR domain of PP5, and both strands of the longest clone were completely sequenced. Glutathione S-Transferase (GST) Fusion Constructs—To make GST fusion constructs, the BamHI/EcoRI fragment of CDC27 (amino acids 295– 823) was cut out from LexA-CDC27(295– 823) and ligated into pGEX-KG to generate GST-CDC27(295– 823). After transfer to nitrocellulose and blocking (in 5% milk, 150 mM NaCl, 50 mM Tris, pH 7.5, 0.02% Tween 20), the membrane was immunoblotted with an anti-VSV-G-tag antibody (P5D4, 1/1,000 dilution), with anti-mouse horseradish peroxidase-conjugated secondary antibody (1/3,000 dilution), and detected by ECL treatment (Amersham). Coverslips were mounted on slides with 100 mM Tris, pH 8.0, 90% glycerol, 1 mg/ml phenylenediamine, containing Hoechst dye 33342 (1 g/ml) to detect DNA
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