calmodulin-dependent protein kinase II in relation to continuous seizure activity

S123

186

VISUALIZATION OF CaMKII ACTIVITY IN THE CYTOPLASM, AND PLASMA MEMBRANE

NAOYUKI INAGAKI’*2, MIWAKO NISHIZAWA’, MIYAMOTO’, A!!D MASAKI INAGAKI’

HIDEYUKI

YAMAMOTD3,

NUCLEUS,

YUSUKE

TAKEUCH13,

EISHICHI

‘Lab. of Biochem., Aichi Cancer Center Res. Inst., Chikusa-ku, Nagoya 464-0021, 2Division of Signal Transduction, Nara Inst. of Science and Technol., Ikoma 630-0101, “Dept. of Pharmacology, Kumamoto Univ. School of Med., Kumamoto 860 Cell signaling is the fundamental strategy by which cells respond to extracellular stimuli and protein kinases play central roles in this process. In general, activation of protein kinases has been analyzed as a whole cell event, because their activities were mostly measured using cell homogenate. However, it has become mcreasingly recognized that Intracellular localization of protein kinase activities is an important factor in conferring specificity and diversity to their signaling networks. Therefore, to unravel the complicated signaling network of protein kinases, it is important to analyze their activity at distinct intracellular sites. The head domain of a cytoskeletal protein vimentin contains a number of phosphorylation sites including Ser82 which is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vivo. The vimentin head domains (VHs) fused with targeting srgnal peptides were expressed in the cytoplasm, plasma membrane, and nucleus of 293 cells. Immunocytochemistry and immunoblot using a site- and phosphorylation-specific antibody MO82 detected VH Ser82 phosphorylations in the cytoplasm, plasma membrane, and nucleus of Ca 2+-stimulated 2Y3 cells. Inhibitor studies and analysis of phosphorylation site specificity confirmed that the VH phosphorylations at Ser82 were induced bv activated CaMKII. Thus, targetable VHs and the antibody MO82 provide a useful tool for investigating CaMKII signaling in subcellular compartments.

187

CA2+/CALMODULlN-DEPENDENT

PROTEIN KINASE II IN RELATION

TO CONTINUOUS

SEIZURE ACTIVITY

YOKO YAMAGATA, Lab. Neurochem,

KUNIHIKO OBATA

Natl. Inst. for Physiol. Sci., Myodaiji,

Ca2+/calmodulin-dependent synaptic plasticity.

Okazaki, 444, Japan

protein kinase II (C&I kinase II) has been implicated in various neuronal functions including

We have been studying its active state in relation to neuronal activity in r,ivo. We previously reported

that acute neuronal excitation induced by electroconvulsive autophosphorylated

treatment in rats caused a transient decrease in the activated,

state of CaM kinase II. In this study, we examined the effect of continuous

by systemic injection of kainic acid into rats. In homogenate after the onset of status epilepticus,

from hippocampus

a profound decrease was observed not only in the Ca2+/calmodulin-independent

activity but also in the total activity of CaM kinase II. We also observed translocation to the particulate fraction.

seizure activity induced

and parietal cortex obtained 30-60 min

of CaM kinase II from the soluble

Thus, continuous seizure activity has profound effect on the active state of CaM kinase II,

which may be related to irreversible neuronal changes associated with continuous seizure activity.

CITRON, A RHO-TARGET,

188

AT GLUTAMATERGIC

TOMOYUKI FURUYASHIKI’,

INTERACTS

SYNAPSES

KAZUKO

WITH PSD-95

IN THE THALAMUS

FUJISAWA’.

AKIKO FUJITA’,

PASCAL MADAULE’.

SHOKO

KIKUMURA’, SHIGEO UCHINO?, MASAYOSHI MISHINA’. HARUHIKO BITO’ and SHUH NARUMNA’ ‘Dept. of Phamacology. Kyoto Univ. Graduate School of Med., ‘Mitsubishi Chemical Corp.. ‘Dept. of Molecular Neurobiology

and Pharmacology,

Tokyo Univ. School of Med.

Proteins of the MAGUK family have recently been shown to be involved in the anchoring and clustering synapses. However, relatively little is known as to whether these multifunctional site for activity-dependent a Rho-effector

and cell type-specific

in the brain, colocalizes

control of the postsynaptic

at many central

scaffold proteins might provide a privileged

signaling machinery.

Here. WC show that Citron,

and associates with PSD-95 both in vivo and in vitro. Elevated Citron expression

found in a predominat population of thalamic excitatory neurons, but not in the principal neurons of the hippocdmpus cerebellum

of the adult mouse brain. Furthermore,

was demonstrated.

a complex consisting

and the

of Citron, PSD-95 and NMDA receptor subunits

Thus Citron may provide a link hetween the Rho signaling cascade and the NMDA receptor-dependent

signaling complex in the thalamus.

was