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Activation of the transcription factor nuclear factor kappa B (NF-[Kappa]B) is controlled by proteolysis of its inhibitory subunit (I[Kappa]B) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of I[Kappa]B[Alpha] by a large multisubunit complex containing I[Kappa]B kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/[Beta]TrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with I[Kappa]B[Alpha] only when I[Kappa]B[Alpha] is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of I[Kappa]B[Alpha] in concert with I[Kappa]B kinases, resulting in nuclear translocation of NF-[Kappa]B. In addition, FWD1 strikingly evoked the ubiquitination of I[Kappa]B[Alpha] in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of I[Kappa]B[Alpha]. These results suggest that the substrate-specific degradation of I[Kappa]B[Alpha] is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated I[Kappa]B[Alpha]. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-[Kappa]B through control of I[Kappa]B protein stability.

Mice lacking the cyclin-dependent kinase (CDK) inhibitor p27(super Kip1) have a large body size, organ hyperplasia and spontaneous pituitary tumors. CDK2 activity increases ten folds in thymocytes lacking p27. The male mice are fertile while females are sterile due to improper ovarian development. The cellular layer pattern development is disrupted in the retinas. However, the lack of the protein has no effect on the inhibitory effect of transforming growth factor beta on the cell cycle or arrest of embryonic fibroblasts in the G1 phase.

Abstract: 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. [ABSTRACT FROM AUTHOR]

The mitochondrial localization of the membrane proteins Bcl-2 and Bcl-xL is essential for their anti-apoptotic function. Here we show that mitochondrial FK506-binding protein 38 (FKBP38), unlike FKBP12, binds to and inhibits calcineurin in the absence of the immunosuppressant FK506, suggesting that FKBP38 is an inherent inhibitor of this phosphatase. FKBP38 is associated with Bcl-2 and Bcl-xL in immunoprecipitation assays and colocalizes with these proteins in mitochondria; in addition, the expression of FKBP38 mutant proteins induces a marked redistribution of Bcl-2 and Bcl-xL. Overexpression of FKBP38 blocks apoptosis, whereas functional inhibition of this protein by a dominant-negative mutant or by RNA interference promotes apoptosis. Thus, FKBP38 might function to inhibit apoptosis by anchoring Bcl-2 and Bcl-xL to mitochondria. [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome pathway plays an important role in control of the abundance of cell cycle regulators. Mice lacking Skp2, an F-box protein and substrate recognition component of an Skpl­Cullin-F-box protein (SCF) ubiquitin ligase, were generated. Although Skp2−/− animals are viable, cells in the mutant mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, and show a reduced growth rate and increased apoptosis. Skp2−/− cells also exhibit increased accumulation of both cyclin E and p27Kip1. The elimination of cyclin E during S and G2 phases is impaired in Skp2−/− cells, resulting in loss of cyclin E periodicity. Biochemical studies showed that Skp2 interacts specifically with cyclin E and thereby promotes its ubiquitylation and degradation both in vivo and in vitro. These results suggest that specific degradation of cyclin E and p27Kip1 is mediated by the SCFSkp2 ubiquitin ligase complex, and that Skp2 may control chromosome replication and centrosome duplication by determining the abundance of cell cycle regulators. [ABSTRACT FROM AUTHOR]

β-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of β-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. β-catenin levels are regulated by the ubiquitin-dependent proteolysis system and β-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3β (GSK-3β)/Axin kinase complex. Here we show that FWD1 (the mouse homologue of Slimb/βTrCP), an F-box/WD40-repeat protein, specifically formed a multi-molecular complex with β-catenin, Axin, GSK-3β and APC. Mutations at the signal-induced phosphorylation site of β-catenin inhibited its association with FWD1. FWD1 facilitated ubiquitination and promoted degradation of β-catenin, resulting in reduced cytoplasmic β-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibited the ubiquitination process and stabilized b-catenin. These results suggest that the Skp1/Cullin/ F-box protein FWD1 (SCFFWD1)-ubiquitin ligase complex is involved in β-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated β-catenin. FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of β-catenin in response to external signals. SCFFWD1 may be critical for tumor development and suppression through regulation of β-catenin protein stability. [ABSTRACT FROM AUTHOR]

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 PP2A B56 phosphatase contributes to the signaling for mTORC1-dependent transcriptional regulation., (© 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)