IGMM, CNRS, Univ Montpellier, Montpellier, France; Equipe labellisée Ligue Nationale Contre le Cancer, Montpellier, France; IGH, CNRS, Univ Montpellier, Montpellier, France.
iBET, Instituto de Biologia Experimental e Tecnologica, Apartado 12, Oeiras, 2781-901, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
J Mol Biol. 2024 Dec 1;436(23):168840. doi: 10.1016/j.jmb.2024.168840. Epub 2024 Oct 26.
The R2TP chaperone is composed of the RUVBL1/RUVBL2 AAA+ ATPases and two adapter proteins, RPAP3 and PIH1D1. Together with HSP90, it functions in the assembly of macromolecular complexes that are often involved in cell proliferation. Here, proteomic experiments using the isolated PIH domain reveals additional R2TP partners, including the Tuberous Sclerosis Complex (TSC) and many transcriptional complexes. The TSC is a key regulator of mTORC1 and is composed of TSC1, TSC2 and TBC1D7. We show a direct interaction of TSC1 with the PIH phospho-binding domain of PIH1D1, which is, surprisingly, phosphorylation independent. Via the use of mutants and KO cell lines, we observe that TSC2 makes independent interactions with HSP90 and the TPR domains of RPAP3. Moreover, inactivation of PIH1D1 or the RUVBL1/2 ATPase activity inhibits the association of TSC1 with TSC2. Taken together, these data suggest a model in which the R2TP recruits TSC1 via PIH1D1 and TSC2 via RPAP3 and HSP90, and use the chaperone-like activities of RUVBL1/2 to stimulate their assembly.
R2TP 伴侣由 RUVBL1/RUVBL2 AAA+ ATP 酶和两个衔接蛋白，RPAP3 和 PIH1D1 组成。它与 HSP90 一起，在组装经常涉及细胞增殖的大分子复合物中发挥作用。在这里，使用分离的 PIH 结构域的蛋白质组学实验揭示了其他 R2TP 伴侣，包括结节性硬化复合物（TSC）和许多转录复合物。TSC 是 mTORC1 的关键调节剂，由 TSC1、TSC2 和 TBC1D7 组成。我们显示 TSC1 与 PIH1D1 的 PIH 磷酸结合结构域之间存在直接相互作用，令人惊讶的是，这种相互作用不依赖于磷酸化。通过使用突变体和 KO 细胞系，我们观察到 TSC2 与 HSP90 和 RPAP3 的 TPR 结构域独立相互作用。此外，PIH1D1 或 RUVBL1/2 ATP 酶活性的失活抑制了 TSC1 与 TSC2 的结合。总之，这些数据表明，R2TP 通过 PIH1D1 招募 TSC1，通过 RPAP3 和 HSP90 招募 TSC2，并利用 RUVBL1/2 的伴侣样活性来刺激它们的组装。
