Phosphorylation of myristoylated alanine-rich protein kinase C substrate by mitogen-activated protein kinase in cultured rat hippocampal neurons following stimulation of glutamate receptors.
Glutamate-induced phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) was investigated in cultured rat hippocampal neurons. In 32P-labeled hippocampal neurons, exposure to 10 microM glutamate induced a long lasting increase in phosphorylation of MARCKS. The long lasting increase in MARCKS phosphorylation mainly required activation of the N-methyl-D-aspartate receptor. Unexpectatively, the MARCKS phosphorylation after the 10-min incubation with glutamate was not inhibited by treatment with calphostin C, a potent inhibitor for protein kinase C (PKC), or down-regulation of PKC but was largely prevented by PD098059, a selective inhibitor for mitogen-activated protein (MAP) kinase kinase. In contrast, the phosphorylation following the short exposure to glutamate was prevented by a combination of PD098059 and calphostin C. The phosphopeptide mapping and immunoblotting analyses confirmed that PKC-dependent phosphorylation of MARCKS was transient and the MAP kinase-dependent phosphorylation was relatively persistent. Investigations of the functional properties also showed that the MARCKS phosphorylation by MAP kinase regulates its calmodulin-binding ability and its interaction with F-actin as seen in the PKC-dependent phosphorylation. These results suggest that glutamate causes a long lasting increase in MARCKS phosphorylation through activation of the N-methyl-D-aspartate receptor and subsequent activation of MAP kinase in the hippocampal neurons.
Ohmitsu
M
M
Department of Pharmacology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan.
Fukunaga
K
K
Yamamoto
H
H
Miyamoto
E
E
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006624:Hippocampus / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009474:Neurons / Q000201:enzymology / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011506:Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017470:Receptors, Glutamate / Q000187:drug effects*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D005419:Flavonoids; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D011506:Proteins; D017470:Receptors, Glutamate; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one