Mitochondria are dynamic organelles that frequently move, divide, and fuse with one another to maintain their architecture and functions. However, the signaling mechanisms involved in these processes are still not well characterized. In this study, we analyze mitochondrial dynamics and morphology in neurons. Using time-lapse imaging, we find that Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1. CaMKIα is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types.

Protein-protein interaction plays important roles in transducing signals elicited from receptors on the cell surface to the nucleus. The Src homology 2 (SH2) and SH3 domains have been shown to bind to tyrosine-phosphorylated proteins (33) and proline-rich motifs (54), respectively (reviewed in references 4, 27, 51, and 61). There are a number of signaling molecules involved in the tyrosine kinase cascade which have either the SH2 domain or the SH3 domain or both, including the GTPase-activating protein for Ras (Ras GAP), phospholipase C-␥, the p85 subunit of PI-3 kinase, and Src and related kinases. While these molecules have enzymatic activities, there is another group of molecules that consist mostly of SH domains without any enzymatic domains. Crk (34, 38), Grb2/Ash (30, 37), Shc (52), and Nck (28) belong to the latter group, the so-called adapter molecules. These multivalent adapter molecules may connect signaling molecules on them. Besides the SH2 and SH3 domains, recently the pleckstrin homology domain also has been identified in a number of signaling molecules (40).The stimulation of cells with various growth factors or cytokines activates their cognate receptor tyrosine kinases or nonreceptor tyrosine kinases associating with the receptors, which results in the tyrosine phosphorylation of various signaling molecules (60). Each tyrosine-phosphorylated molecule is recognized and bound by a specific SH2 domain (64, 65). Ras GAP, phospholipase C-␥, and the p85 subunit of PI-3 kinase have been shown to become tyrosine phosphorylated and bind to activated receptors through their SH2 domains (4,27,51,61). The interaction between SH3 domains and proline-rich motifs seems to be constitutive and not to depend on stimulation of the cells (54). These interactions by the SH2 and SH3 domains are responsible for the formation of multimolecular signaling complexes, some of which translocate to the plasma membrane.

The worldwide prevalence of type 2 diabetes (T2D), which is caused by a combination of environmental and genetic factors, is increasing. With regard to genetic factors, variations in the gene encoding Cdk5 regulatory associated protein 1-like 1 (Cdkal1) have been associated with an impaired insulin response and increased risk of T2D across different ethnic populations, but the molecular function of this protein has not been characterized. Here, we show that Cdkal1 is a mammalian methylthiotransferase that biosynthesizes 2-methylthio-N 6 -threonylcarbamoyladenosine (ms 2 t 6 A) in tRNA Lys (UUU) and that it is required for the accurate translation of AAA and AAG codons. Mice with pancreatic β cell-specific KO of Cdkal1 (referred to herein as β cell KO mice) showed pancreatic islet hypertrophy, a decrease in insulin secretion, and impaired blood glucose control. In Cdkal1-deficient β cells, misreading of Lys codon in proinsulin occurred, resulting in a reduction of glucosestimulated proinsulin synthesis. Moreover, expression of ER stress-related genes was upregulated in these cells, and abnormally structured ER was observed. Further, the β cell KO mice were hypersensitive to high fat diet-induced ER stress. These findings suggest that glucose-stimulated translation of proinsulin may require fully modified tRNA Lys (UUU), which could potentially explain the molecular pathogenesis of T2D in patients carrying cdkal1 risk alleles.

Oxytocin is an essential hormone for mammalian labor and lactation. Here, we show a new function of oxytocin in causing plastic changes in hippocampal synapses during motherhood. In oxytocin-perfused hippocampal slices, one-train tetanus stimulation induced long-lasting, long-term potentiation (L-LTP) and phosphorylation of cyclic AMP-responsive element binding protein (CREB), and MAP kinase inhibitors blocked these inductions. An increase in CREB phosphorylation and L-LTP induced by one-train tetanus were observed in the multiparous mouse hippocampus without oxytocin application. Furthermore, intracerebroventricular injection of oxytocin in virgin mice improved long-term spatial learning in vivo, whereas an injection of oxytocin antagonist in multiparous mice significantly inhibited the improved spatial memory, L-LTP and CREB phosphorylation. These findings indicate that oxytocin is critically involved in improving hippocampus-dependent learning and memory during motherhood in mice.

Upon screening of a human hippocampus library with a bovine Nck5a cDNA, we uncovered a distinct clone encoding a 39-kDa isoform of Nck5a. The isoform, designated the neuronal Cdk5 activator isoform (p39 nck5ai ), showed a high degree of sequence similarity to p35 nck5a with 57% amino acid identity. Northern blot analysis detected its mRNA transcript in bovine and rat cerebrum and cerebellum, but not in any other rat tissues examined. In situ hybridization showed that Nck5ai was enriched in CA1 to CA3 of the hippocampus, but absent in the fimbria of hippocampal formation. Among seven cell lines in proliferating cultures, only PC12 and N2A, two cell lines capable of differentiating into neuron-like cells, were found to contain Nck5ai mRNA. A 30-kDa truncated form of Nck5ai expressed as a glutathione S-transferase fusion protein in Escherichia coli] was found to associate with Cdk5 to form an active Cdk5 kinase. Thus, the isoform shares many common characteristics with p35 nck5a , including Ckd5 activating activity and brain-and neuronspecific expression. Both proteins show limited sequence homology to cyclins, suggesting that they define a new family of cyclin-dependent kinase-activating proteins.The cell division cycle gene, cdc2, in fission yeast performs rate-limiting functions in both G 1 /S and G 2 /M transitions. The protein product of the gene, p34 cdc2

Galanin is a widely distributed neuropeptide with a variety of physiological functions. Three galanin receptor subtypes, GALR1, GALR2, and GALR3, have been reported. We isolated a novel galanin-like peptide (GALP) from porcine hypothalamus by observing its activity for increasing [35 S]GTP␥S binding to a membrane preparation of GALR2-transfected cells. The peptide had 60 amino acid residues and a non-amidated C terminus. The amino acid sequence of GALP-(9 -21) was completely identical to that of galanin-(1-13). A cloned porcine GALP cDNA indicated that GALP was processed from a 120-amino acid GALP precursor protein. The structures of rat and human GALP-(1-60) were deduced from cloned cDNA, which indicated that the amino acid sequences 1-24 and 41-53 were highly conserved between humans, rats, and pigs. Receptor binding studies revealed that porcine GALP-(1-60) had a high affinity for the GALR2 receptor (IC 50 ‫؍‬ 0.24 nM) and a lower affinity for the GALR1 receptor (IC 50 ‫؍‬ 4.3 nM). In contrast, galanin showed high affinity for the GALR1 (IC 50 ‫؍‬ 0.097 nM) and GALR2 receptors (IC 50 ‫؍‬ 0.48 nM). GALP is therefore an endogenous ligand that preferentially binds the GALR2 receptor, whereas galanin is relatively non-selective.Galanin, a C-terminally amidated peptide with 29 amino acid residues (non-amidated peptide with 30 residues in humans) was originally isolated from porcine intestine (1) and was later found to be ubiquitous in the central and peripheral nervous systems. It exerts diverse regulatory functions including central modulation of cognition, nociception, and feeding behavior, endocrine control of pituitary and pancreatic hormones, and regulation of gastrointestinal smooth muscle contractions (for review, see Refs. 2, 3).Structurally, galanin is unrelated to any known family of neuropeptides or regulatory peptides, suggesting the presence of unknown members of a galanin peptide family. Indeed, the existence of a galanin-like peptide(s) in mammalian tissues has been proposed for several reasons. First, molecular heterogeneity of galanin-like immunoreactivity has been reported by several groups. Rökaeus et al. (4) first reported that rat brain and ileum contained galanin-like peptides that cross-reacted with galanin antiserum but differed from galanin in chromatographic characterization. Nevertheless, some of these immunoreactivities may be the result of galanin precursors or galaninderived peptides, as described in the subsequent studies (5). Recently, Wang et al. (6) re-evaluated the existence of a novel galanin-like peptide in rat islets by showing the presence of galanin-like immunoreactivity that cross-reacted with antibody against porcine galanin but did not with antibody against rat galanin. Second, the physiological function of the three galanin receptor subtypes, GALR1 1 (7, 8), GALR2 (9, 10), and GALR3 (11,12), is unlikely to be solely the distribution of different signals to target cells. The low affinity of human galanin for human GALR3 (12) suggests the possibility that these receptor s...

Calcineurin and calpain, a Ca2؉ /calmodulin-dependent protein phosphatase and a Ca 2؉ -dependent cysteine protease, respectively, mediate neuronal cell death through independent cascades. Here, we report that during neuroexcitotoxicity, calcineurin A (CnA) is directly cleaved by calpain in vitro and in vivo, resulting in the enzyme being converted to an active form. Mass spectrometry identified three cleavage sites in CnA, two of which were constitutively active forms. Overexpression of the cleaved CnA induced caspase activity and neuronal cell death. Calpain inhibitors and membranepermeable calpastatin peptides not only blocked the cleavage of CnA, but also protected against excitotoxic neuronal cell death in vitro and in vivo. These results indicate that CnA is a crucial target for calpain, and the calpain-mediated activation of CnA triggers excitotoxic neurodegeneration. This study established a molecular link between calpain and calcineurin, thereby demonstrating a new mechanism for proteolytical regulation of calcineurin by calpain in response to certain pathological states.

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation–induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with β-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.