Atsuko Ohtake

Activation of Protein-tyrosine Phosphatase SH-PTP2 by a Tyrosine-based Activation Motif of a Novel Brain Molecule

BIT (a rain mmunoglobulin-like molecule with yrosine-based activation motifs) is a brain-specific membrane protein which has two cytoplasmic TAMs (yrosine-based ctivation otifs). Using the Far Western blotting technique, we detected association of a 70-kDa protein with the tyrosine-phosphorylated TAMs of BIT. A mouse brain cDNA library in λgt11 was screened for this association, and two positive clones encoding tyrosine phosphatase SH-PTP2 were isolated. SH-PTP2 has two SH2 domains and is believed to function as a positive mediator in receptor tyrosine kinase signaling. SH-PTP2 and BIT were coimmunoprecipitated from phosphorylated rat brain lysate, and BIT was a major tyrosine-phosphorylated protein associated with SH-PTP2 in this lysate. This interaction was also observed in Jurkat T cells transfected with BIT cDNA depending on tyrosine phosphorylation of BIT. Bisphosphotyrosyl peptides corresponding to BIT-TAMs stimulated SH-PTP2 activity 33-35-fold in vitro, indicating that two SH2 domains of SH-PTP2 simultaneously interact with two phosphotyrosines of BIT-TAM. Our findings suggest that the tyrosine phosphorylation of BIT results in stimulation of the signal transduction pathway promoted by SH-PTP2 and that BIT is probably a major receptor molecule in the brain located just upstream of SH-PTP2.

Imaging of cAMP‐dependent protein kinase activity in living neural cells using a novel fluorescent substrate

In order to visualize the activity of the cAMP‐dependent protein kinase (PKA) in living cells, we have constructed a new fluorescence PKA substrate by conjugating a fluorescence probe to a partial amino acid sequence of PKA regulatory domain II which contains a specific autophosphorylation site. The fluorescent peptide was cell‐permeable and became phosphorylated when the intracellular cAMP concentration was increased, resulting in a decrease in its fluorescence intensity. In NG108‐15 cells, PKA activity was localized to the cytosol around the nucleus. In cultured hippocampal neurons, addition of l‐glutamate caused PKA activation associated with increase of the cellular cAMP.

Tau-tubulin kinase phosphorylates tau at Ser-208 and Ser-210, sites found in paired helical filament-tau

Hyperphosphorylated tau protein is known to be a major component of the paired helical filaments (PHFs) that accumulate in the brain of Alzheimers patients. The kinase that phosphorylated Ser‐208 and Ser‐210 in PHF‐tau had remained unknown. We used anti‐pS208 and anti‐pS210 antibodies and Western blots to confirm that the tau‐tubulin kinase (TTK) phosphorylates tau at Ser‐208 and at Ser‐210. Using partial amino acid sequences of purified bovine brain TTK, a mouse cDNA of TTK was isolated and the sequence was determined. Its 963 bp coding region is composed of 320 amino acids and encodes a 36 kDa protein indistinguishable in size from authentic bovine brain TTK. Our immunoblot analysis demonstrated that TTK is ubiquitously distributed in the rat tissues, and that it is developmentally regulated in the rat brain.

A novel tau-tubulin kinase from bovine brain

During purification of tau protein kinase I and II from the bovine brain extract, a new tau protein kinase was detected and purified with phosphocellulose, gel filtration, S‐Sepharose and AF‐Heparin column chromatography. The molecular mass of the enzyme was determined to be 32 kDa by gel filtration and activity staining on SDS‐PAGE. The enzyme is a Ser/Thr protein kinase phosphorylating tau, β‐tubulin, MAP2 and α‐casein. Employing many synthetic peptides, the recognition site of this enzyme appears to be ‐SR‐. The enzyme requires no second messenger and is inhibited with high concentration of heparin, but not by inhibitors of CKI. These results indicate that this enzyme, tau‐tubulin kinase is novel and distinct from TPKI, II and CKI, II.

Binding of chimeric analogs of ω-conotoxin MVIIA and MVIIC to the N- and P/Q-type calcium channels

Despite their high sequence homology, the peptide neurotoxins ω‐conotoxin MVIIA and MVIIC selectively block N‐ and P/Q‐type calcium channels, respectively. To study the recognition mechanism of calcium channel subtypes, two chimeric analogs of ω‐conotoxin MVIIA and MVIIC were synthesized by exchanging their N‐ and C‐terminal halves. Binding assay for both N‐ and P/Q‐type calcium channels showed that amino acid residues restricted to the N‐terminal half are important for the recognition of N‐type channels, whereas essential residues for P/Q‐type channel recognition are widely spread over the whole ω‐conotoxin molecule.

Familial Alzheimer's disease-associated mutations block translocation of full-length presenilin 1 to the nuclear envelope

A polyclonal antibody, M5, to the hydrophilic loop domain of human presenilin 1 (PS1) was prepared. Western blot and immunoprecipitation analyses showed that M5 specifically recognized the processed C-terminal fragment, but not the full-length PS1. Epitope mapping analysis revealed that the essential sequence for recognition of the C-terminal fragment by M5 is DPEAQRR (302-308). The recognition of the C-terminal fragment by M5 in a processing-dependent manner was further confirmed by competitive enzyme-linked immunosorbent assay using the synthetic peptide L281 (281-311), which contains the putative processing site and the preceding amino acids to the site. Although L281 contains the epitope sequence for M5, the maximum inhibition was only 14%. Immunocytochemistry using M5 combined with hL312, which recognizes both full-length PS1 and the C-terminal fragment, allowed us to distinguish the localization of the processed C-terminal fragment from that of full-length PS1. Confocal microscopy demonstrated that the full-length form of wild-type PS1 is preferentially located in the nuclear envelope, while the processed C-terminal fragment is mainly present in the endoplasmic reticulum (ER). However, PS1 with familial Alzheimers disease-associated mutations could not translocate to the nuclear envelope, and both the full-length and processed mutants were co-localized in the ER.

Binding of Ala-scanning analogs of ω-conotoxin MVIIC to N- and P/Q-type calcium channels

ω‐Conotoxin MVIIC binds to P/Q‐type calcium channels with high affinity and N‐type channels with low affinity. To reveal the residues essential for subtype selectivity, we synthesized Ala‐scanning analogs of MVIIC. Binding assays using rat cerebellar P2 membranes suggested that Thr11, Tyr13 and Lys2 are essential for binding to both N‐ and P/Q‐type channels, whereas Lys4 and Arg22 are important for binding to P/Q‐type channels. These results suggest that MVIIC interacts with P/Q‐type channels via a large surface, in good agreement with previous observations using chimeric analogs.

Imaging of Ca2+/calmodulin-dependent protein kinase II activity in hippocampal neurones

Our aim was to visualize the dynamic features of Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. In order to do so, we synthesized a new reagent by conjugating a fluoroprobe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), to syntide 2, a specific peptide substrate for CaMKII. In cell-free conditions, the conjugate was found to be an effective indicator of calmodulin activation by Ca2+ and the subsequent activation of CaMKII. The reagent is cell-permeable and can stain living cells when bath-applied. Using this technique we were able to obtain fluorescence images of stained cells and analyse the dynamic features of CaMKII inside the cells by means of image processing. Regional heterogeneity of CaMKII activation in cultured hippocampal neurones was seen following L-glutamate administration.

Extracellular carbohydrate-signal triggering cAMP-dependent protein kinase-dependent neuronal actin-reorganization.

Cell surface glycoconjugates are thought to mediate cell-cell recognition and to play roles in neuronal development and functions. We demonstrated here that exposure of neuronal cells to nanomolar levels of glyco-chains with an N-acetylgalactosamine (GalNAc) residue at the non-reducing termini (GalNAc-S) such as GalNAcbeta4(Neu5Acalpha3)Galbeta4GlcCer (GM2) ganglioside, its oligosaccharide portion, GalNAcbeta4Galbeta4GlcCer (Gg(3)) Cer, GalNAcalpha3GalNAcbeta3Galalpha4Galbeta4GlcCer (Gb(5)) Cer (Forssman hapten) and alpha1-4 linked oligomers of GalNAc, induced a rapid and transient activation of cAMP-dependent protein kinase (PKA) in subplasmalemma. The treatment was accompanied by peripheral actin polymerization and filopodia formation in NG108-15 cells and primary cultured hippocampal neurons, but not in glial cells. A cAMP-dependent protein kinase (PKA) selective inhibitor and an adenylate cyclase inhibitor blocked both PKA activation and the subsequent filopodia formation. A small GTPase cdc42 was a potential downstream target of GalNAc-S-activated PKA. These results suggest that extracellular GalNAc-S serve as potential regulators of the filopodia formation in neuronal cells by triggering the activation of PKA followed by cdc42 up-regulation via a cell surface receptor-like component. Filopodia formation induced by GalNAc-S may have a physiological relevance because long-term exposure to GalNAc-S enhanced F-actin-rich dendrite generation of primary cultured hippocampal neurons, and PKA-dependent dendritic outgrowth and branch formation of primary cultured cerebellar Purkinje neurons, in which actin isoforms were localized to motile structures in dendrites. These findings provide evidence for a novel GalNAc/PKA-signaling cascade in regulating some neuronal maturation.

Role of Thr11 in the binding of ω-conotoxin MVIIC to N-type Ca2+ channels

As replacement of Thr11 of ω‐conotoxin MVIIC with Ala significantly reduced the affinity for both N‐ and P/Q‐type calcium channels, we examined the effect of substitution at this position with other residues. Binding assays using rat cerebellar P2 membranes showed that the affinity is in the order of Leu>Val, aminobutyric acid, Thr>Asn≫Ser, Ala, Asp, Phe, Tyr for N‐type channels and Thr>Leu, Val, aminobutyric acid, Asn, Ser>Ala≫Asp, Phe, Tyr for P/Q‐type channels, suggesting that aliphatic amino acids with longer side chains are favorable for block of N‐type channels. The effects of substitution were examined electrophysiologically in BHK cells expressing N‐type Ca2+ channels. Inhibition of Ba2+ current by the analogs did not completely correlate with binding affinity, although binding to BHK cells was comparable to rat cerebellar membranes.