Masayuki Matsushita

Regulation of IgA production by naturally occurring TNF/iNOS-producing dendritic cells

Immunoglobulin-A has an irreplaceable role in the mucosal defence against infectious microbes. In human and mouse, IgA-producing plasma cells comprise ∼20% of total plasma cells of peripheral lymphoid tissues, whereas more than 80% of plasma cells produce IgA in mucosa-associated lymphoid tissues (MALT). One of the most biologically important and long-standing questions in immunology is why this ‘biased’ IgA synthesis takes place in the MALT but not other lymphoid organs. Here we show that IgA class-switch recombination (CSR) is impaired in inducible-nitric-oxide-synthase-deficient (iNOS-/-; gene also called Nos2) mice. iNOS regulates the T-cell-dependent IgA CSR through expression of transforming growth factor-β receptor, and the T-cell-independent IgA CSR through production of a proliferation-inducing ligand (APRIL, also called Tnfsf13) and a B-cell-activating factor of the tumour necrosis factor (TNF) family (BAFF, also called Tnfsf13b). Notably, iNOS is preferentially expressed in MALT dendritic cells in response to the recognition of commensal bacteria by toll-like receptor. Furthermore, adoptive transfer of iNOS+ dendritic cells rescues IgA production in iNOS-/- mice. Further analysis revealed that the MALT dendritic cells are a TNF-α/iNOS-producing dendritic-cell subset, originally identified in mice infected with Listeria monocytogenes. The presence of a naturally occurring TNF-α/iNOS-producing dendritic-cell subset may explain the predominance of IgA production in the MALT, critical for gut homeostasis.

CaM kinase Iα–induced phosphorylation of Drp1 regulates mitochondrial morphology

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.

Characterization of the Mechanism of Regulation of Ca2+/ Calmodulin-dependent Protein Kinase I by Calmodulin and by Ca2+/Calmodulin-dependent Protein Kinase Kinase

Ca2+/calmodulin-dependent protein kinase I (CaMKI) is maintained in an autoinhibited state by the interaction of a COOH-terminal helix-loop-helix (Ile286-Met316) regulatory domain with the catalytic core. Activation of the enzyme by calmodulin (CaM) also allows CaMKI to be phosphorylated and activated by a second enzyme, CaMK kinase (CaMKK). To more thoroughly characterize the regulation of CaMKI by CaM and its interrelationship with phosphorylation by CaMKK, we have carried out a detailed structure-function analysis using recombinant wild-type (WT) and mutant forms of CaMKI and CaMKK. CaMKI-WT, in the absence of CaM, or CaMKI-299 and CaMKI-298 were autoinhibited and could not be phosphorylated by CaMKK-433 (a truncated constitutively active form of CaMKK). Removal of Phe298(CaMK-297) generated a constitutively active form of CaMKI that was also phosphorylated by CaMKK-433. CaMKI-WT was essentially inactive in the absence of CaM (K 0.5 for activation by CaM ∼30 nm). Mutation of Ile294 and Phe298 to alanine (CaMKI-2A) resulted in measurable basal enzyme activity. Additional mutation of Ile286 and Val290 to alanine (CaMKI-4A) increased this basal activity. Mutation of Trp303 (CaMKI-W303S) resulted in a large increase in the K 0.5 for CaM (∼100 μm), supporting a role for this residue as an initial target for CaM. Mutation of Phe307 (CaMKI-F307A) resulted in increased basal enzyme activity, supporting a role for this residue in autoinhibition of CaMKI. Together these studies demonstrate the critical role of specific amino acids in the autoinhibition of CaMKI and also in its activation by CaM and phosphorylation by CaMKK.

Inhibition of the Ca2+/calmodulin-dependent protein kinase I cascade by cAMP-dependent protein kinase.

Several recent studies have shown that Ca2+/calmodulin-dependent protein kinase I (CaMKI) is phosphorylated and activated by a protein kinase (CaMKK) that is itself subject to regulation by Ca2+/calmodulin. In the present study, we demonstrate that this enzyme cascade is regulated by cAMP-mediated activation of cAMP-dependent protein kinase (PKA). In vitro, CaMKK is phosphorylated by PKA and this is associated with inhibition of enzyme activity. The major site of phosphorylation is threonine 108, although additional sites are phosphorylated with lower efficiency. In vitro, CaMKK is also phosphorylated by CaMKI at the same sites as PKA, suggesting that this regulatory phosphorylation might play a role as a negative-feedback mechanism. In intact PC12 cells, activation of PKA with forskolin resulted in a rapid inhibition of both CaMKK and CaMKI activity. In hippocampal slices CaMKK was phosphorylated under basal conditions, and activation of PKA led to an increase in phosphorylation. Two-dimensional phosphopeptide mapping indicated that activation of PKA led to increased phosphorylation of multiple sites including threonine 108. These results indicate that in vitro and in intact cells the CaMKK/CaMKI cascade is subject to inhibition by PKA-mediated phosphorylation of CaMKK. The phosphorylation and inhibition of CaMKK by PKA is likely to be involved in modulating the balance between cAMP- and Ca2+-dependent signal transduction pathways.

Inhibition of Tumor Necrosis Factor Signal Transduction in Endothelial Cells by Dimethylaminopurine

Tumor necrosis factor (TNF) promotes diverse responses in endothelial cells that are important to the host response to infections and malignancies; however, less is known of the postreceptor events important to TNF action in endothelial cells than in many other cell types. Since phosphorylation cascades are implicated in cytokine signaling, the effects of the protein kinase inhibitor dimethylaminopurine (DMAP) on TNF action in bovine aortic endothelial cells (BAEC) were investigated. In BAEC, TNF promotes phosphorylation of eukaryotic initiation factor 4E (eIF-4E), c-Jun N-terminal kinase (JNK) and ceramide-activated protein kinase activities, Jun-b expression, prostacyclin production, and, when protein synthesis is inhibited, cytotoxicity. DMAP abrogated or significantly attenuated each of these responses to TNF, without affecting the specific binding of TNF to its receptors. Histamine, another agent active in the endothelium, promotes phosphorylation of elongation factor-2 (EF-2) and prostacyclin production, but not phosphorylation of eIF-4E in BAEC. Histamine-stimulated EF-2 phosphorylation was not inhibited and prostacyclin production was unaffected by DMAP. These observations demonstrate that a distinct signal transduction cascade, which can be selectively inhibited by DMAP, promotes the response of BAEC to TNF. Thus, we have identified a reagent, DMAP, that may be useful for characterizing the TNF signal transduction pathway.

Recent advances in protein transduction technology.

During the past 15 years, a variety of peptides, known as protein transduction domains (PTDs), or cell-penetrating peptides (CPPs), have been characterized for their ability to translocate into live cells. There are now numerous examples of biologically active full-length proteins and peptides that have been successfully delivered to cells and tissues, both in vitro and in vivo. One of the principal mechanisms of protein transduction is via electrostatic interactions with the plasma membrane, subsequent penetration into the cells by macropinocytosis, and release into the cytoplasm and nuclei by retrograde transport. Recent reports have also now shown that some of the limitations of protein transduction technology have been overcome. In particular, the use of ubiquitination-resistant proteins has been demonstrated to be a more effective strategy for transduction because the half-life of these molecules is significantly increased. Moreover, the use of the NH2-terminal domain of the influenza virus hemagglutinin-2 subunit (HA2) or photosensitive PTDs has been shown to specifically enhance macropinosome escape. Hence, these and other recent advances in protein transduction technologies have created a number of possibilities for the development of new peptide-based drugs.

OGFOD1, a member of the 2-oxoglutarate and iron dependent dioxygenase family, functions in ischemic signaling

The 2‐oxoglutarate and iron dependent dioxygenase family are crucial for cellular adaptation to changes in oxygen concentration. We found that cells with OGFOD1 gene silencing in this family showed resistance to cell death under ischemia, and cDNA microarray analysis of OGFOD1 knockout human cells revealed downregulation of ATPAF1. Although reintroduction of the OGFOD1 wild‐type gene to OGFOD1 KO cells restored ATPAF1 mRNA levels, the catalytically inactive OGFOD1 mutants did not. Furthermore, introduction of ATPAF1 gene to OGFOD1 KO cells induced ischemic cell death. Thus, OGFOD1 plays an important role in ischemic cell survival and an OGFOD1 iron binding residue is required for ATPAF1 gene expression.

Involvement of calcineurin in glutamate-induced mitochondrial dynamics in neurons

Alterations in the morphology and movement of mitochondria influence neuronal viability. However, the precise mechanisms of such alterations are unclear. In this study, we showed calcineurin was involved in the regulation of mitochondrial dynamics. Glutamate stimulation inhibited mitochondrial movement and decreased mitochondrial length in neurons. FK506 and cyclosporine A, calcineurin inhibitors, attenuated the effects of glutamate on mitochondrial dynamics. It was also found that glutamate treatment dephosphorylated, a proapoptotic protein, Bad and promoted its translocation to mitochondria in neurons via calcineurin. These results provide important new insights into intracellular signaling pathways that regulate mitochondrial dynamics and neuronal cell death.

Transformation of eEF1Bδ into heat‐shock response transcription factor by alternative splicing

Protein translation factors have crucial roles in a variety of stress responses. Here, we show that eukaryotic elongation factor 1Bδ (eEF1Bδ) changes its structure and function from a translation factor into a heat‐shock response transcription factor by alternative splicing. The long isoform of eEF1Bδ (eEF1BδL) is localized in the nucleus and induces heat‐shock element (HSE)‐containing genes in cooperation with heat‐shock transcription factor 1 (HSF1). Moreover, the amino‐terminal domain of eEF1BδL binds to NF‐E2‐related factor 2 (Nrf2) and induces stress response haem oxygenase 1 (HO1). Specific inhibition of eEF1BδL with small‐interfering RNA completely inhibits Nrf2‐dependent HO1 induction. In addition, eEF1BδL directly binds to HSE oligo DNA in vitro and associates with the HSE consensus in the HO1 promoter region in vivo. Thus, the transcriptional role of eEF1BδL could provide new insights into the molecular mechanism of stress responses.

Forebrain-specific constitutively active CaMKKα transgenic mice show deficits in hippocampus-dependent long-term memory

The Ca(2+)/calmodulin (CaM) kinase cascade is activated by Ca(2+) influx through the voltage-dependent Ca(2+) channels and the NMDA receptor. CaM kinase kinase (CaMKK), the most upstream kinase of the CaM kinase cascade, phosphorylates and activates both CaM kinase I (CaMKI) and CaMKIV, resulting in activation of cyclic AMP-responsive element binding protein (CREB)-dependent gene transcription. Using transgenic techniques, we created mutant mice in which a constitutively active form of CaMKK1, the autoinhibitory domain truncated protein, is over-expressed specifically in the forebrain. In these mice, although performance was normal in basal activity and short-term memory, specific impairments were shown in hippocampus-dependent long-term memory after training in spatial memory tasks and after contextual fear conditioning. In cultured neurons of these mice, phosphorylation of CaMKI was significantly increased in basal states, whereas the activity range of CaMKI phosphorylation by brain-derived neurotrophic factor (BDNF) and KCl stimulation was significantly diminished in mutant mice. Our results define a critical role for CaMKKα in synaptic plasticity and the retention of hippocampus-dependent long-term memory.