Michiko Shirane

Targeted disruption of Skp2 results in accumulation of cyclin E and p27Kip1, polyploidy and centrosome overduplication

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 Skp1–Cullin–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.

An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin.

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 β‐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.

Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis

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.

Down-regulation of p27(Kip1) by two mechanisms, ubiquitin-mediated degradation and proteolytic processing.

The intracellular level of p27 Kip1 , a cyclin-dependent kinase (CDK) inhibitory protein, is rapidly reduced at the G1/S transition phase when the cell cycle pause ceases. In this study, we demonstrated that two posttranslational mechanisms were involved in p27 Kip1 breakdown: degradation via the ubiquitin (Ub)-proteasome pathway and proteolytic processing that rapidly eliminates the cyclin-binding domain. We confirmed that p27 Kip1 was ubiquitinated in vitroas well as in vivo. The p27 Kip1 -ubiquitination activity was higher at the G1/S boundary than during the G0/G1 phase, and p27 Kip1 ubiquitination was reduced significantly when the lysine residues at positions 134, 153, and 165 were replaced by arginine, suggesting that these lysine residues are the targets for Ub conjugation. In parallel with its Ub-dependent degradation, p27 Kip1 was processed rapidly at its N terminus, reducing its molecular mass from 27 to 22 kDa, by a ubiquitination-independent but adenosine triphosphate (ATP)-dependent mechanism with higher activity during the S than the G0/G1 phase. This 22-kDa intermediate had no cyclin-binding domain at its N terminus and virtually no CDK2 kinase inhibitory activity. These results suggest that p27 Kip1 is eliminated by two independent mechanisms, ubiquitin-mediated degradation and ubiquitin-independent processing, during progression from the G1 to S phase.

Common Pathway for the Ubiquitination of IκBα, IκBβ, and IκBε Mediated by the F-Box Protein FWD1

FWD1 (the mouse homolog of DrosophilaSlimb and Xenopus βTrCP, a member of the F-box- and WD40 repeat-containing family of proteins, and a component of the SCF ubiquitin ligase complex) was recently shown to interact with IκBα and thereby to promote its ubiquitination and degradation. This protein has now been shown also to bind to IκBβ and IκBε as well as to induce their ubiquitination and proteolysis. FWD1 was shown to recognize the conserved DSGΨXS motif (where Ψ represents the hydrophobic residue) present in the NH2-terminal regions of these three IκB proteins only when the component serine residues are phosphorylated. However, in contrast to IκBα and IκBβ, the recognition site in IκBε for FWD1 is not restricted to the DSGΨXS motif; FWD1 also interacts with other sites in the NH2-terminal region of IκBε. Substitution of the critical serine residues in the NH2-terminal regions of IκBα, IκBβ, and IκBε with alanines also markedly reduced the extent of FWD1-mediated ubiquitination of these proteins and increased their stability. These data indicate that the three IκB proteins, despite their substantial structural and functional differences, all undergo ubiquitination mediated by the SCFFWD1 complex. FWD1 may thus play an important role in NF-κB signal transduction through regulation of the stability of multiple IκB proteins.

Down-Regulation of p27Kip1 Expression Is Required for Development and Function of T Cells

The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation. The cyclin-dependent kinase inhibitor p27Kip1 is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4−CD8− thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27Kip1 expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4+CD8+, CD4+CD8−, and CD4−CD8+ stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27Kip1 expression. The development of thymocytes in the transgenic strain in which p27Kip1 is most abundant (p27-Tghigh mice) appeared to be blocked at the CD4−CD8−CD25+CD44low stage. Peripheral T cells from p27-Tghigh mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells. Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tghigh mice. These results suggest that down-regulation of p27Kip1 expression is required for the development, proliferation, and immunoresponsiveness of T cells.

Protrudin serves as an adaptor molecule that connects KIF5 and its cargoes in vesicular transport during process formation

Protrudin is a key regulator of vesicular transport during neurite extension. Using a proteomics approach, this study identified KIF5 as a protrudin-associated protein. Protrudin functioned synergistically with KIF5 and facilitated the interaction of KIF5 with Rab11, suggesting that the Rab11–protrudin–KIF5 complex contributes to vesicular transport in neurons.

Selective escape of proteins from the mitochondria during mitophagy

Mitophagy refers to the degradation of mitochondria by the autophagy system that is regulated by Parkin and PINK1, mutations in the genes for which have been linked to Parkinsons disease. Here we show that certain mitochondrial outer membrane proteins, including FKBP38 and Bcl-2, translocate from the mitochondria to the endoplasmic reticulum (ER) during mitophagy, thereby escaping degradation by autophagosomes. This translocation depends on the ubiquitylation activity of Parkin and on microtubule polymerization. Photoconversion analysis confirmed that FKBP38 detected at the ER during mitophagy indeed represents preexisting protein transported from the mitochondria. The escape of FKBP38 and Bcl-2 from the mitochondria is determined by the number of basic amino acids in their COOH-terminal signal sequences. Furthermore, the translocation of FKBP38 is essential for the suppression of apoptosis during mitophagy. Our results thus show that not all mitochondrial proteins are degraded during mitophagy, with some proteins being evacuated to the ER to prevent unwanted apoptosis.

Anchoring of the 26S proteasome to the organellar membrane by FKBP38

FK506‐binding protein 38 (FKBP38) is a member of the immunophilin family that resides in the mitochondrial outer membrane and the endoplasmic reticulum (ER) membrane. To investigate the physiological function of FKBP38, we performed a comprehensive search for proteins with which it interacts in human cells by liquid chromatographic and mass spectrometric analysis of FKBP38 immunoprecipitates. Almost all subunits of the 26S proteasome were thus found to interact with FKBP38. In vivo co‐immunoprecipitation analyses confirmed that FKBP38 indeed associates with the 26S proteasome via its three tandem tetratricopeptide repeats (TPRs). Binding assays in vitro also revealed that FKBP38 directly interacts with the S4 subunit of the 19S proteasome. Immunofluorescence analysis demonstrated that the subcellular distributions of FKBP38 and the 26S proteasome partially overlapped at mitochondria. Both the abundance and activity of the proteasome in a membrane fraction were markedly reduced for mouse embryonic fibroblasts prepared from Fkbp38−/– mice compared with those prepared from wild‐type mice. These results suggest that FKBP38 functions to anchor the 26S proteasome at the organellar membrane.

Promotion of neurite extension by protrudin requires its interaction with vesicle-associated membrane protein-associated protein.

Protrudin is a protein that contains a Rab11-binding domain and a FYVE (lipid-binding) domain and that functions to promote neurite formation through interaction with the GDP-bound form of Rab11. Protrudin also contains a short sequence motif designated FFAT (two phenylalanines in an acidic tract), which in other proteins has been shown to mediate binding to vesicle-associated membrane protein-associated protein (VAP). We now show that protrudin associates and colocalizes with VAP-A, an isoform of VAP expressed in the endoplasmic reticulum. Both the interaction between protrudin and VAP-A as well as the induction of process formation by protrudin were markedly inhibited by mutation of the FFAT motif. Furthermore, depletion of VAP-A by RNA interference resulted in mislocalization of protrudin as well as in inhibition of neurite outgrowth induced by nerve growth factor in rat pheochromocytoma PC12 cells. These defects resulting from depletion of endogenous rat VAP-A in PC12 cells were corrected by forced expression of (RNA interference-resistant) human VAP-A but not by VAP-A mutants that have lost the ability to interact with protrudin. These results suggest that VAP-A is an important regulator both of the subcellular localization of protrudin and of its ability to stimulate neurite outgrowth.