Ollendorff V, Donoghue D J
Department of Chemistry and Biochemistry and the Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0367, USA.
J Biol Chem. 1997 Dec 19;272(51):32011-8. doi: 10.1074/jbc.272.51.32011.
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.
进化上保守的多亚基复合物，即环体或后期促进复合物，参与催化M期多种底物的泛素化，使其被26S蛋白酶体降解并完成有丝分裂。后期促进复合物的八个亚基中的三个（CDC16、CDC23和CDC27）已被证明在M期被磷酸化，并且它们的磷酸化是后期促进复合物作为泛素连接酶发挥活性所必需的。后期促进复合物的几个亚基含有四肽重复序列，这是一种参与蛋白质/蛋白质相互作用的蛋白质基序。PP5是一种丝氨酸/苏氨酸磷酸酶，也含有四个四肽重复序列基序拷贝。在这里，我们通过双杂交分析和体外结合相结合的方法表明，PP5与后期促进复合物的两个亚基CDC16和CDC27相互作用。这种物理相互作用仅需要PP5的NH2末端结构域，该结构域包含所有四个四肽重复序列。缺失分析表明，与PP5结合的位点定位于CDC16和CDC27中四肽重复序列的COOH末端区域。此外，间接免疫荧光显示PP5定位于有丝分裂纺锤体装置。PP5与CDC16和CDC27的直接相互作用，以及其在有丝分裂中与纺锤体重叠的定位，表明PP5可能参与后期促进复合物活性的调节。
