Hirokazu Nakatsumi

Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

ABSTRACT D-type cyclins play a pivotal role in G1-S progression of the cell cycle, and their expression is frequently deregulated in cancer. Cyclin D1 has a half-life of only ∼30 min as a result of its ubiquitylation and proteasomal degradation, with various F-box proteins, including Fbxo4, Fbxw8, Skp2, and Fbxo31, having been found to contribute to its ubiquitylation. We have now generated Fbxo4-deficient mice and found no abnormalities in these animals. Cyclin D1 accumulation was thus not observed in Fbxo4−/− mouse tissues. The half-life of cyclin D1 in mouse embryonic fibroblasts (MEFs) prepared from Fbxo4−/−, Fbxw8−/−, and Fbxo4−/−; Fbxw8−/− mice also did not differ from that in wild-type MEFs. Additional depletion of Skp2 and Fbxo31 in Fbxo4−/−; Fbxw8−/− MEFs by RNA interference did not affect cyclin D1 stability. Although Fbxo31 depletion in MEFs increased cyclin D1 abundance, this effect appeared attributable to upregulation of cyclin D1 mRNA. Furthermore, abrogation of the function of the Skp1–Cul1–F-box protein (SCF) complex or the anaphase-promoting complex/cyclosome (APC/C) complexes did not alter the half-life of cyclin D1, whereas cyclin D1 degradation was dependent largely on proteasome activity. Our genetic analyses thus do not support a role for any of the four F-box proteins examined in cyclin D1 degradation during normal cell cycle progression. They suggest the existence of other ubiquitin ligases that target cyclin D1 for proteolysis.

A large-scale targeted proteomics assay resource based on an in vitro human proteome

Targeted proteomics approaches are of value for deep and accurate quantification of protein abundance. Extending such methods to quantify large numbers of proteins requires the construction of predefined targeted assays. We developed a targeted proteomics platform—in vitro proteome–assisted multiple reaction monitoring (MRM) for protein absolute quantification (iMPAQT)—by using >18,000 human recombinant proteins, thus enabling protein absolute quantification on a genome-wide scale. Our platform comprises experimentally confirmed MRM assays of mass tag (mTRAQ)-labeled peptides to allow for rapid and straightforward measurement of the absolute abundance of predefined sets of proteins by mass spectrometry. We applied iMPAQT to delineate the quantitative metabolic landscape of normal and transformed human fibroblasts. Oncogenic transformation gave rise to relatively small but global changes in metabolic pathways resulting in aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. iMPAQT should facilitate quantitative biology studies based on protein abundance measurements.

Structural basis for the assembly of the Ragulator-Rag GTPase complex

The mechanistic target of rapamycin complex 1 (mTORC1) plays a central role in regulating cell growth and metabolism by responding to cellular nutrient conditions. The activity of mTORC1 is controlled by Rag GTPases, which are anchored to lysosomes via Ragulator, a pentameric protein complex consisting of membrane-anchored p18/LAMTOR1 and two roadblock heterodimers. Here we report the crystal structure of Ragulator in complex with the roadblock domains of RagA-C, which helps to elucidate the molecular basis for the regulation of Rag GTPases. In the structure, p18 wraps around the three pairs of roadblock heterodimers to tandemly assemble them onto lysosomes. Cellular and in vitro analyses further demonstrate that p18 is required for Ragulator-Rag GTPase assembly and amino acid-dependent activation of mTORC1. These results establish p18 as a critical organizing scaffold for the Ragulator-Rag GTPase complex, which may provide a platform for nutrient sensing on lysosomes.mTORC1 activity is controlled through Rag GTPases, which are anchored to the lysosome through the Ragulator. Here, the authors give molecular insights into Ragulator-Rag GTPase assembly and present the crystal structures of the Ragulator alone and in complex with the RagA-C roadblock domains.

Nuclear-cytoplasmic shuttling protein PP2AB56 contributes to mTORC1-dependent dephosphorylation of FOXK1

Mammalian target of rapamycin complex 1 (mTORC1) kinase is a master regulator of the cellular response to nutrition‐related signals such as insulin and amino acids. mTORC1 is activated on the lysosomal membrane and induces phosphorylation of a variety of downstream molecules. We previously showed that activated mTORC1 induces protein phosphatase 2A (PP2A)‐mediated dephosphorylation of the transcription factor forkhead box K1 (FOXK1). The mechanism underlying the signal transduction from the cytoplasmic mTORC1 to the nuclear FOXK1 has remained unclear, however, we now show that a nuclear–cytoplasmic transport system is necessary for the mTORC1‐FOXK1 signal transduction. This reaction is mediated by a shuttling protein B56, which is a regulatory subunit of PP2A and plays an essential role in the mTORC1‐dependent dephosphorylation of FOXK1. These results suggest that PP2AB56 phosphatase contributes to the signaling for mTORC1‐dependent transcriptional regulation.