Song Changcheng, Wang Qing, Li Chou-Chi H
Basic Research Program, SAIC-Frederick, National Cancer Institute, Frederick, Maryland 21702, USA.
J Biol Chem. 2003 Feb 7;278(6):3648-55. doi: 10.1074/jbc.M208422200. Epub 2002 Nov 20.
The 97-kDa valosin-containing protein (p97-VCP) plays a role in a wide variety of cellular activities, many of which are regulated by the ubiquitin-proteasome (Ub-Pr)-mediated degradation pathway. We previously demonstrated that VCP binds to multi-ubiquitin chains and may act as a molecular chaperone that targets the ubiquitinated substrates to the proteasome for degradation. In this report, we show that although the ubiquitin chain-binding activity, carried out by the N-terminal 200 residues (N domain), is necessary for the degradation of proteasome substrates, it is not sufficient. Using in vitro degradation assays, we demonstrated that the entire VCP molecule, consisting of the N domain and two ATPase domains D1 and D2, is required for mediating the Ub-Pr degradation. The ATPase activity of VCP requires Mg(2+), and is stimulated by high temperature. Under optimal conditions, VCP hydrolyzes ATP with a K(m) of approximately 0.33 mm and a V(max) of approximately 0.52 nmol P(i) min(-1) microg(-1). At a physiological temperature, mutation in D2 significantly inhibits the ATPase activity, while that in D1 has little effect. Interestingly, mutations in D1, but not D2, abolish the heat-stimulated ATPase activity. Thus, we provide the first demonstration that the ATPase activity of VCP is required for mediating the Ub-Pr degradation, that D2 accounts for the major ATPase activity, and that D1 contributes to the heat-induced activity.
97千道尔顿含缬酪肽蛋白（p97-VCP）在多种细胞活动中发挥作用，其中许多活动受泛素-蛋白酶体（Ub-Pr）介导的降解途径调控。我们之前证明VCP与多泛素链结合，可能作为一种分子伴侣，将泛素化底物靶向蛋白酶体进行降解。在本报告中，我们表明，虽然由N端200个残基（N结构域）执行的泛素链结合活性对于蛋白酶体底物的降解是必需的，但并不充分。通过体外降解试验，我们证明由N结构域以及两个ATP酶结构域D1和D2组成的整个VCP分子对于介导Ub-Pr降解是必需的。VCP的ATP酶活性需要Mg(2+)，并受高温刺激。在最佳条件下，VCP水解ATP的K(m)约为0.33 mM，V(max)约为0.52 nmol Pi min(-1) μg(-1)。在生理温度下，D2中的突变显著抑制ATP酶活性，而D1中的突变影响很小。有趣的是，D1中的突变而非D2中的突变消除了热刺激的ATP酶活性。因此，我们首次证明VCP的ATP酶活性对于介导Ub-Pr降解是必需的，D2负责主要的ATP酶活性，而D1促成热诱导活性。
