Yuji Funakoshi

Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3′ end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.

NMDA Receptor-Mediated PIP5K Activation to Produce PI(4,5)P2 Is Essential for AMPA Receptor Endocytosis during LTD

NMDA receptor activation leads to clathrin-dependent endocytosis of postsynaptic AMPA receptors. Although this process controls long-term depression (LTD) induction in the hippocampus, how it is regulated by neuronal activities is not completely clear. Here, we show that Ca²⁺ influx through the NMDA receptor activates calcineurin and protein phosphatase 1 to dephosphorylate phosphatidylinositol 4-phosphate 5-kinaseγ661 (PIP5Kγ661), the major phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-producing enzyme in the brain. Bimolecular fluorescence complementation analysis revealed that the dephosphorylated PIP5Kγ661 became associated with the clathrin adaptor protein complex AP-2 at postsynapses in situ. NMDA-induced AMPA receptor endocytosis and low-frequency stimulation-induced LTD were completely blocked by inhibiting the association between dephosphorylated PIP5Kγ661 and AP-2 and by overexpression of a kinase-dead PIP5Kγ661 mutant in hippocampal neurons. Furthermore, knockdown of PIP5Kγ661 inhibited the NMDA-induced AMPA receptor endocytosis. Therefore, NMDA receptor activation controls AMPA receptor endocytosis during hippocampal LTD by regulating PIP5Kγ661 activity at postsynapses.

Anti‐proliferative protein Tob negatively regulates CPEB3 target by recruiting Caf1 deadenylase

Tob is a member of the anti‐proliferative protein family, which functions in transcription and mRNA decay. We have previously demonstrated that Tob is involved in the general mechanism of mRNA decay by mediating mRNA deadenylation through interaction with Caf1 and a general RNA‐binding protein, PABPC1. Here, we focus on the role of Tob in the regulation of specific mRNA. We show that Tob binds directly to a sequence‐specific RNA‐binding protein, cytoplasmic polyadenylation element‐binding protein 3 (CPEB3). CPEB3 negatively regulates the expression of a target by accelerating deadenylation and decay of its mRNA, which it achieves by tethering to the mRNA. The carboxyl‐terminal RNA‐binding domain of CPEB3 binds to the carboxyl‐terminal unstructured region of Tob. Tob then binds Caf1 deadenylase and recruits it to CPEB3 to form a ternary complex. The CPEB3‐accelerated deadenylation was abrogated by a dominant‐negative mutant of either Caf1 or Tob. Together, these results indicate that Tob mediates the recruitment of Caf1 to the target of CPEB3 and elicits deadenylation and decay of the mRNA. Our results provide an explanation of how Tob regulates specific biological processes.

Phospholipase D signalling and its involvement in neurite outgrowth

Since the original discovery and structural elucidation of the mammalian phospholipase D (PLD), its potential to play a role in the lipid signalling pathway has attracted considerable interest. Now, it is generally accepted that different PLD isozymes are likely to serve diverse functions in membrane trafficking, endocytosis, exocytosis, cell growth, differentiation and actin cytoskeletal organization. In addition, PLDs are known to play a key role in neurite outgrowth, especially axon outgrowth, in neuronal cells.

Regulation of PIP5K activity by Arf6 and its physiological significance

The phospholipid kinase phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) catalyzes the phosphorylation of the membrane phospholipid phosphatidylinositol 4‐phosphate to generate the pleiotropic phospholipid phosphatidylinositol 4,5‐bisphosphate [PI(4,5)P2]. To date, three mammalian PIP5K isozymes, α, β, and γ, and several splicing variants of the γ isozyme have been identified. These PIP5K isozymes and PIP5Kγ variants play critical roles in various cellular functions through their product PI(4,5)P2. The small GTPase Arf6 is one of the key activators of PIP5K. Increasing evidence suggests that PIP5K functions as a downstream effector of Arf6 to regulate a wide variety of cellular functions, such as exocytosis, endocytosis, endosomal recycling, membrane ruffle formation, immune response, and bacterial invasion. In this review, we place our focus on the recent advances in Arf6/PIP5K signaling and its linkage to cellular functions. J. Cell. Physiol. 226: 888–895, 2011.

Molecular Mechanisms of N-Formyl-Methionyl-Leucyl-Phenylalanine-Induced Superoxide Generation and Degranulation in Mouse Neutrophils: Phospholipase D Is Dispensable

ABSTRACT Phospholipase D (PLD), which produces the lipid messenger phosphatidic acid (PA), has been implicated in superoxide generation and degranulation in neutrophils. The basis for this conclusion is the observation that primary alcohols, which interfere with PLD-catalyzed PA production, inhibit these neutrophil functions. However, off-target effects of primary alcohols cannot be totally excluded. Here, we generated PLD−/− mice in order to reevaluate the involvement of PLD in and investigate the molecular mechanisms of these neutrophil functions. Surprisingly, N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced these functions in PLD−/− neutrophils, and these functions were suppressed by ethanol. These results indicate that PLD is dispensable for these neutrophil functions and that ethanol nonspecifically inhibits them, warning against the use of primary alcohols as specific inhibitors of PLD-mediated PA formation. The calcium ionophore ionomycin and the membrane-permeative compound 1-oleoyl-2-acetyl-sn-glycerol (OADG) synergistically induced superoxide generation. On the other hand, ionomycin alone induced degranulation, which was further augmented by OADG. These results demonstrate that conventional protein kinase C (cPKC) is crucial for superoxide generation, and a Ca2+-dependent signaling pathway(s) and cPKC are involved in degranulation in mouse neutrophils.

The scaffold protein JIP3 functions as a downstream effector of the small GTPase ARF6 to regulate neurite morphogenesis of cortical neurons

MINT‐7892353, MINT‐7892615, MINT‐7892657, MINT‐7892672, MINT‐7892549, MINT‐7892738: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JIP3 (uniprotkb:Q9ESN9) by anti tag coimmunoprecipitation (MI:0007)

Arf6 regulates tumour angiogenesis and growth through HGF-induced endothelial β1 integrin recycling

Anti-angiogenic drugs targeting vascular endothelial cell growth factor receptor have provided modest clinical benefit, in part, owing to the actions of additional angiogenic factors that stimulate tumour neoangiogenesis in parallel. To overcome this redundancy, approaches targeting these other signalling pathways are required. Here we show, using endothelial cell-targeted mice, that the small GTPase Arf6 is required for hepatocyte growth factor (HGF)-induced tumour neoangiogenesis and growth. Arf6 deletion from endothelial cells abolishes HGF-stimulated β1 integrin recycling. Pharmacological inhibition of the Arf6 guanine nucleotide exchange factor (GEF) Grp1 efficiently suppresses tumour vascularization and growth. Grp1 as well as other Arf6 GEFs, such as GEP100, EFA6B and EFA6D, regulates HGF-stimulated β1 integrin recycling. These findings provide insight into the mechanism of HGF-induced tumour angiogenesis and offer the possibility that targeting the HGF-activated Arf6 signalling pathway may synergize with existing anti-angiogenic drugs to improve clinical outcomes.

Antiproliferative protein Tob directly regulates c-myc proto-oncogene expression through cytoplasmic polyadenylation element-binding protein CPEB

The regulation of mRNA deadenylation constitutes a pivotal mechanism of the post-transcriptional control of gene expression. Here we show that the antiproliferative protein Tob, a component of the Caf1–Ccr4 deadenylase complex, is involved in regulating the expression of the proto-oncogene c-myc. The c-myc mRNA contains cis elements (CPEs) in its 3′-untranslated region (3′-UTR), which are recognized by the cytoplasmic polyadenylation element-binding protein (CPEB). CPEB recruits Caf1 deadenylase through interaction with Tob to form a ternary complex, CPEB–Tob–Caf1, and negatively regulates the expression of c-myc by accelerating the deadenylation and decay of its mRNA. In quiescent cells, c-myc mRNA is destabilized by the trans-acting complex (CPEB–Tob–Caf1), while in cells stimulated by the serum, both Tob and Caf1 are released from CPEB, and c-Myc expression is induced early after stimulation by the stabilization of its mRNA as an ‘immediate-early gene’. Collectively, these results indicate that Tob is a key factor in the regulation of c-myc gene expression, which is essential for cell growth. Thus, Tob appears to function in the control of cell growth at least, in part, by regulating the expression of c-myc.

Tissue- and development-dependent expression of the small GTPase Arf6 in mice

The small GTPase Arf6 is a member of the Arf (ADP‐ribosylation factor) family. Although the function of Arf6 has been heavily studied at the cellular level, its physiological function at the whole animal level is largely unknown. In this study, we examined both the tissue distribution and developmental timing of Arf6 expression in wild type mice to obtain valuable information to speculate on the physiological function of Arf6. Western blot analysis using anti‐Arf6 antibody revealed that Arf6 was ubiquitously expressed with its developmental timing differing in a tissue‐specific manner. These results were supported by Arf6 mRNA in situ hybridization experiments, which showed that Arf6 was highly expressed in the polarized epithelial cells and embryonic mesenchymal cells of most tissues in a temporally dependent manner. Taken in toto, our results suggest that the expression of Arf6 in mouse tissues is precisely regulated in a development‐ and tissue‐dependent manner. Developmental Dynamics 239:3416–3435, 2010.