Yoshiyuki Yoshimura

Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography‐tandem mass spectrometry

Protein constituents of the postsynaptic density (PSD) fraction were analysed using an integrated liquid chromatography (LC)‐based protein identification system, which was constructed by coupling microscale two‐dimensional liquid chromatography (2DLC) with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and an automated data analysis system. The PSD fraction prepared from rat forebrain was solubilized in 6 m guanidium hydrochloride, and the proteins were digested with trypsin after S‐carbamoylmethylation under reducing conditions. The tryptic peptide mixture was then analysed with the 2DLC‐MS/MS system in a data‐dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database for protein identification. In triplicate analyses, the system allowed assignments of 5264 peptides, which could finally be attributed to 492 proteins. The PSD contained various proteins involved in signalling transduction, including receptors, ion channel proteins, protein kinases and phosphatases, G‐protein and related proteins, scaffold proteins, and adaptor proteins. Structural proteins, including membrane proteins involved in cell adhesion and cell–cell interaction, proteins involved in endocytosis, motor proteins, and cytoskeletal proteins were also abundant. These results provide basic data on a major protein set associated with the PSD and a basis for future functional studies of this important neural machinery.

Enhancement by streptozotocin of O−2 radical generation by the xanthine oxidase system of pancreatic β-cells

Spin‐trapping techniques and electron spin resonance (ESR) spectroscopy were used to study the relationship between the effect of streptozotocin (STZ) on pancreatic β‐cells and free radical formation by these cells. Results showed that STZ enhanced generation of the DMPO‐OH radical adduct, which is a degradation product of the superoxide anion (O− 2) in the presence of cellular components, in a hypoxanthine‐xanthine oxidase (XOD) system with a homogenate of β‐cells. This enhancing effect was also observed in a system without cellular components; STZ increased the signal height due to the O− 2 radical in a concentration‐dependent manner and caused a maximum of 150% enhancement at a concentration of 1.5 mM. Thus, STZ seemed to enhance the generation of the O− 2 radical in the XOD system, probably by some mechanism of its interaction with XOD. Pancreatic β‐cells exhibited a high XOD activity and a very low superoxide dismutase activity. Therefore, the present result supports the possibility that the cytotoxic effect of STZ is closely related to free radical generation in pancreatic β‐cells.

Investigation of protein substrates of Ca2+/calmodulin-dependent protein kinase II translocated to the postsynaptic density

To elucidate the physiological significance of the translocation of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II), we investigated substrates of CaM kinase II in the postsynaptic density (PSD). PSD proteins were phosphorylated by CaM kinase II of its PSD complex, and separated by two-dimensional gel electrophoresis. More than 28 proteins were phosphorylated under experimental conditions. Proteins corresponding to CaM kinase II substrates were excised from the gels, eluted electrophoretically, and then sequenced. Several substrates were identified, including PSD95, SAP90, alpha-internexin, neurofilament L chain, cAMP phosphodiesterase, and alpha- and beta-tubulin. Some substrates were also identified by immunoblotting, including N-methyl-D-aspartic acid (NMDA) receptor 2B subunit, 1-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor 1 (GluR1), neurofilament H chain and dynamin. PSD95, SAP90, dynamin, and alpha-internexin were demonstrated for the first time to be substrates of CaM kinase II. NMDA receptor 2B subunit and GluR1 existed as major substrates in the PSD. Moreover, translocation of CaM kinase II was inhibited by phosphorylation of PSD proteins. These results suggest that CaM kinase II plays important roles in the regulation of synaptic functions through phosphorylation of PSD proteins.

Interaction of Arc with CaM kinase II and stimulation of neurite extension by Arc in neuroblastoma cells expressing CaM kinase II

We investigated the relationship between Arc (activity-regulated cytoskeleton-associated protein) and Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). Arc and CaM kinase II were concentrated in the postsynaptic density. These proteins were accumulated after electroconvulsive treatment. Arc increased about 2.5-fold within 30 min and was maintained at this level for 8h after the stimulation. CaM kinase II also increased within 30 min and remained at this level for at least 24h. The interaction of Arc with CaM kinase II was demonstrated using GST-Arc fusion protein, and confirmed in neuroblastoma cells by immunoprecipitation. We examined the function of Arc by introducing Arc cDNA into neuroblastoma cells expressing CaM kinase II. The cells expressing both Arc and CaM kinase II had longer neurites than those expressing CaM kinase II alone. Arc itself did not promote neurite outgrowth. The growth of neurites by Arc was completely blocked by treatment with KN62, an inhibitor of CaM kinases. These results indicated that Arc potentiated the action of CaM kinase II for neurite extension.

Protein phosphatase 1 is involved in the dissociation of Ca2+/calmodulin-dependent protein kinase II from postsynaptic densities

Autophosphorylation‐dependent translocation of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) to postsynaptic densities (PSDs) from cytosol may be a physiologically important process during synaptic activation. We investigated a protein phosphatase responsible for dephosphorylation of the kinase. CaM kinase II was shown to be targeted to two sites using the gel overlay method in two‐dimensional gel electrophoresis. Protein phosphatase 1 (PP1) was identified to dephosphorylate CaM kinase II from its complex with PSDs using phosphatase inhibitors and activators, and purified phosphatases. The kinase was released from PSDs after its dephosphorylation by PP1.

Phosphorylation-dependent Reversible Association of Ca2+/Calmodulin-dependent Protein Kinase II with the Postsynaptic Densities

The association of soluble Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) with postsynaptic densities (PSDs) was determined by activity assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting of the enzyme. Soluble CaM kinase II was autophosphorylated with ATP in the presence of Ca2+and calmodulin, and then it was incubated with PSDs. Autophosphorylated CaM kinase II was precipitated with PSDs by centrifugation. The kinase that was not autophosphorylated did not precipitate with PSDs. These results indicate that the soluble previously autophosphorylated CaM kinase II associates with PSDs and forms PSD-CaM kinase II complex. A maximum of about 60 μg of soluble CaM kinase II bound to 1 mg of PSD protein under the experimental conditions. Ca2+-independent activity generated by autophosphorylation of the kinase was retained in the PSD-CaM kinase II complex. The CaM kinase II thus associated with PSDs phosphorylated a number of PSD proteins in both the absence and presence of Ca2+. When the CaM kinase II-PSD complex was incubated at 30 °C, its Ca2+-independent activity was gradually decreased. This decrease was correlated with dephosphorylation of the kinase and its release from PSD-CaM kinase II complex. These results indicate that CaM kinase II reversibly translocates to PSDs in a phosphorylation-dependent manner.

Phosphorylation of tau protein to sites found in Alzheimer's disease brain is catalyzed by Ca2+/calmodulin-dependent protein kinase II as demonstrated tandem mass spectrometry

Neuronal Ca2+/calmodulin-dependent protein kinase II (CaMKII) is one of the most abundant protein kinases in the brain, and phosphorylates a broad range of substrate proteins. The phosphorylation of microtubule tau by CaMKII was investigated using tandem mass spectrometry (MS/MS). Recombinant human tau was phosphorylated at Thr212, Ser214, Ser262, and Ser356 by CaMKII. The phosphorylation of these sites is found in paired helical filament (PHF)-tau. In addition to these sites, Ser131 and Thr135 were phosphorylated by CaMKII. Phosphorylation at Ser131, Thr135, Thr212 and Ser214 by CaMKII has not been reported previously. Thr212 and Ser214 are in the consensus phosphorylation sequence of CaMKII (RXXS/T), and non-fetal-type phosphorylation sites of tau. Non-fetal-type phosphorylation may produce PHF-tau. These results suggested that CaMKII is involved in the phosphorylation of tau in Alzheimers disease brain.

Interaction of LDL receptor-related protein 4 (LRP4) with postsynaptic scaffold proteins via its C-terminal PDZ domain-binding motif, and its regulation by Ca2+/calmodulin-dependent protein kinase II

We cloned here a full‐length cDNA of Dem26[ Tian et al. (1999)Mol. Brain Res., 72, 147–157], a member of the low‐density lipoprotein (LDL) receptor gene family from the rat brain. We originally named the corresponding protein synaptic LDL receptor‐related protein (synLRP) [ Tian et al. (2002) Soc. Neurosci. Abstr., 28, 405] and have renamed it LRP4 to accord it systematic nomenclature (GenBankTM accession no. AB073317). LRP4 protein interacted with postsynaptic scaffold proteins such as postsynaptic density (PSD)‐95 via its C‐terminal tail sequence, and associated with N‐methyl‐d‐aspartate (NMDA)‐type glutamate receptor subunit. The mRNA of LRP4 was localized to dendrites, as well as somas, of neuronal cells, and the full‐length protein of 250 kDa was highly concentrated in the brain and localized to various subcellular compartments in the brain, including synaptic fractions. Immunocytochemical study using cultured cortical neurons suggested surface localization in the neuronal cells both in somas and dendrites. Ca2+/calmodulin‐dependent protein kinase II (CaMKII) phosphorylated the C‐terminal cytoplasmic region of LRP4 at Ser1887 and Ser1900, and the phosphorylation at the latter site suppressed the interaction of the protein with PSD‐95 and synapse‐associated protein 97 (SAP97). These findings suggest a postsynaptic role for LRP4, a putative endocytic multiligand receptor, and a mechanism in which CaMKII regulates PDZ‐dependent protein–protein interactions and receptor dynamics.

Involvement of Protein Kinase Cδ/Extracellular Signal-regulated Kinase/Poly(ADP-ribose) Polymerase-1 (PARP-1) Signaling Pathway in Histamine-induced Up-regulation of Histamine H1 Receptor Gene Expression in HeLa Cells

The histamine H1 receptor (H1R) gene is up-regulated in patients with allergic rhinitis. However, the mechanism and reason underlying this up-regulation are still unknown. Recently, we reported that the H1R expression level is strongly correlated with the severity of allergic symptoms. Therefore, understanding the mechanism of this up-regulation will help to develop new anti-allergic drugs targeted for H1R gene expression. Here we studied the molecular mechanism of H1R up-regulation in HeLa cells that express H1R endogenously in response to histamine and phorbol 12-myristate 13-acetate (PMA). In HeLa cells, histamine stimulation caused up-regulation of H1R gene expression. Rottlerin, a PKCδ-selective inhibitor, inhibited up-regulation of H1R gene expression, but Go6976, an inhibitor of Ca2+-dependent PKCs, did not. Histamine or PMA stimulation resulted in PKCδ phosphorylation at Tyr311 and Thr505. Activation of PKCδ by H2O2 resulted in H1R mRNA up-regulation. Overexpression of PKCδ enhanced up-regulation of H1R gene expression, and knockdown of the PKCδ gene suppressed this up-regulation. Histamine or PMA caused translocation PKCδ from the cytosol to the Golgi. U0126, an MEK inhibitor, and DPQ, a poly(ADP-ribose) polymerase-1 inhibitor, suppressed PMA-induced up-regulation of H1R gene expression. These results were confirmed by a luciferase assay using the H1R promoter. Phosphorylation of ERK and Raf-1 in response to PMA was also observed. However, real-time PCR analysis showed no inhibition of H1R mRNA up-regulation by a Raf-1 inhibitor. These results suggest the involvement of the PKCδ/ERK/poly(ADP-ribose) polymerase-1 signaling pathway in histamine- or PMA-induced up-regulation of H1R gene expression in HeLa cells.

Overexpression of α and β isoforms of Ca2+/calmodulin-dependent protein kinase II in neuroblastoma cells — H-7 promotes neurite outgrowth

Since the α and β isoforms of CaM kinase II are known to be expressed almost exclusively in the brain, we compared the effect of overexpression of the β isoform of CaM kinase II with that of the α isoform. The subcellular distribution of the α isoform was different from that of the β isoform, although the catalytic properties of the α and β isoforms expressed in transfected cells were similar to those of brain CaM kinase II. The α isoform was found in the soluble fraction more than in the particulate fraction, whereas most of the β isoform bound to subcellular structures. In the cell overexpressing α and β isoforms of CaM kinase II, neurite extension was promoted when compared with the morphology of neo transfectants. Neurite outgrowth of cells overexpressing CaM kinase II was further stimulated by the treatment of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a selective but not absolutely specific inhibitor of protein kinase C. The morphological change was rapid and observed within 1 h followed by H-7 treatment. Morphological changes, such as the number of cells with neurites and length of neurites were greater in the β cells than in the α cells. Chelerythrine, a specific inhibitor of protein kinase C, also stimulated the neurite outgrowth of these cells. Some substrates of CaM kinase II related to neurite outgrowth were detected in cells overexpressing CaM kinase II stimulated with H-7. These results suggest that CaM kinase II and protein kinase C play an important role in the control of cell change, and that the subcellular distribution of CaM kinase II is important for regulating cellular functions efficiently.