Targeted gene disruption studies have established that the c-Jun NH2-terminal kinase (JNK) is required for the stress-induced release of mitochondrial cytochrome c and apoptosis, and that the Bax subfamily of Bcl-2-related proteins is essential for JNK-dependent apoptosis. However, the mechanism by which JNK regulates Bax has remained unsolved. Here we demonstrate that activated JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3, a cytoplasmic anchor of Bax. Phosphorylation of 14-3-3 led to dissociation of Bax from this protein. Expression of phosphorylation-defective mutants of 14-3-3 blocked JNK-induced Bax translocation to mitochondria, cytochrome c release and apoptosis. Collectively, these results have revealed a key mechanism of Bax regulation in stress-induced apoptosis

Akt is a common mediator of cell survival in a variety of circumstances. Although some candidate Akt targets have been described, the function of Akt is not fully understood, particularly because of the cell type-and context-dependent apoptosis regulation. In this study, we demonstrate that one of the mechanisms by which Akt antagonizes apoptosis involves the inhibition of Nur77, a transcription factor implicated in T-cell receptor-mediated apoptosis. It has been suggested that Akt phosphorylates Nur77 directly, but whether Akt suppresses biological functions of Nur77 remains unknown. We found that Akt inhibited the DNA binding activity of Nur77 and stimulated its association with 14-3-3 in a phosphorylation site-dependent manner. Moreover, we found that expression of Akt suppressed Nur77-induced apoptosis in fibroblasts and activation-induced cell death of T-cell hybridomas. The inhibition of Nur77 by Akt suggests a mechanism that explains how T-cell receptor activation can promote survival in some instances even when Nur77 is induced. Collectively, these results may suggest that Akt is a negative regulator of Nur77 in T-cell apoptosis.

Current proteomic studies clarified canonical synaptic proteins that are common to many types of synapses. However, proteins of diversified functions in a subset of synapses are largely hidden because of their low abundance or structural similarities to abundant proteins. To overcome this limitation, we have developed an “ultra-definition” (UD) subcellular proteomic workflow. Using purified synaptic vesicle (SV) fraction from rat brain, we identified 1,466 proteins, three times more than reported previously. This refined proteome includes all canonical SV proteins, as well as numerous proteins of low abundance, many of which were hitherto undetected. Comparison of UD quantifications between SV and synaptosomal fractions has enabled us to distinguish SV-resident proteins from potential SV-visitor proteins. We found 134 SV residents, of which 86 are present in an average copy number per SV of less than one, including vesicular transporters of nonubiquitous neurotransmitters in the brain. We provide a fully annotated resource of all categorized SV-resident and potential SV-visitor proteins, which can be utilized to drive novel functional studies, as we characterized here Aak1 as a regulator of synaptic transmission. Moreover, proteins in the SV fraction are associated with more than 200 distinct brain diseases. Remarkably, a majority of these proteins was found in the low-abundance proteome range, highlighting its pathological significance. Our deep SV proteome will provide a fundamental resource for a variety of future investigations on the function of synapses in health and disease.

Background: Rab-type small GTPases are conserved membrane trafficking proteins in all eukaryotes. Results: Knockdown of Rab17 in mouse hippocampal neurons results in a marked reduction in the number of dendritic branches and total dendrite length. Conclusion: Rab17 regulates dendritic morphogenesis and postsynaptic development in hippocampal neurons. Significance: Our findings reveal the first molecular link between membrane trafficking and dendritogenesis.

Background: Small GTPase Rab17 regulates dendritic morphogenesis of hippocampal neurons, but its activation mechanism is completely unknown. Results: Expression of Rabex-5 in mouse hippocampal neurons promoted dendritic localization of Rab17, and Rabex-5 knockdown resulted in inhibition of neurite morphogenesis. Conclusion: Rabex-5 regulates neurite morphogenesis by activating Rab5 and Rab17. Significance: Our findings revealed a crucial role of Rabex-5 and its targets in neuronal development.

The yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca 2؉ -permeable nonselective cation channel composed of 548 amino acid residues. A physiological role of the Mid1 channel is known to maintain the viability of yeast cells exposed to mating pheromone, but its structural basis remains to be clarified. To solve this problem, we identified the mutation sites of mid1 mutant alleles generated by in vivo ethyl methanesulfonate mutagenesis and found that two mid1 alleles have nonsense mutations at the codon for Trp 441 , generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn 389 to Thr 548 . In vitro random mutagenesis with hydroxylamine also showed that the carboxyl-terminal region is essential. To identify the functional portion of the carboxyl-terminal region in detail, we performed a progressive carboxyl-terminal truncation followed by functional analyses and found that the truncated protein produced from the mid1 allele bearing the amber mutation at the codon for Phe 522 (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca 2؉ uptake activity to a wild-type level, whereas N521Am did not. This result indicates that the carboxyl-terminal domain spanning from Asn 389 to Asn 521 is required for Mid1 function. Interestingly, this domain is cysteine-rich, and alaninescanning mutagenesis revealed that seven out of 10 cysteine residues are unexchangeable. These results clearly indicate that the carboxyl-terminal domain including the cysteine residues is important for Mid1 function.

Efficient retrieval of the synaptic vesicle (SV) membrane from the presynaptic plasma membrane, a process called endocytosis, is crucial for the fidelity of neurotransmission, particularly during sustained neural activity. Although multiple modes of endocytosis have been identified, it is clear that the efficient retrieval of the major SV cargos into newly formed SVs during any of these modes is fundamental for synaptic transmission. It is currently believed that SVs are eventually reformed via a clathrin-dependent pathway. Various adaptor proteins recognize SV cargos and link them to clathrin, ensuring the efficient retrieval of the cargos into newly formed SVs. Here, we summarize our current knowledge of the molecular signatures within individual SV cargos that underlie efficient retrieval into SV membranes, as well as discuss possible contributions of the mechanisms under physiological conditions.

The double C2 (Doc2) family is characterized by an N-terminal Munc13-1-interacting domain and C-terminal tandem C2 domains, and it comprises three isoforms, Doc2alpha, Doc2beta, and Doc2gamma, in humans and mice. Doc2alpha, the best-characterized, brain-specific isoform, exhibits Ca(2+)-dependent phospholipid-binding activity through its C2A domain, and the Ca(2+)-binding activity is thought to be important for the regulation of Ca(2+)-dependent exocytosis. In contrast to the C2A domain, however, nothing is known about the physiological functions of the C2B domain in regulated exocytosis. In this study, we demonstrated by a mutation analysis that the polybasic sequence in the C2B domain of Doc2alpha (306 KKSKHKTCVKKK 317) is required for binding of syntaxin-1a/synaptosome-associated protein of 25 kDa (SNAP-25) heterodimer. We also investigated the effect of Lys-to-Gln (named KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in PC12 cells. A Doc2alpha(KQ) mutant, which lacks binding activity toward syntaxin-1a/SNAP-25 heterodimer, significantly decreased the number of plasma membrane-docked vesicles before stimulation and strongly inhibited high-KCl-induced exocytosis from the plasma membrane-docked vesicles. These results indicate that the polybasic sequence in the C2B domain functions as a binding site for syntaxin-1a/SNAP-25 heterodimer and controls the number of 'readily releasable' vesicles in neuroendocrine cells.