Rho-associated kinase phosphorylates MARCKS in human neuronal cells.
Myristoylated alanine-rich C kinase substrate (MARCKS) is a filamentous actin bundling protein and has multiple sites for phosphorylation, by which the biochemical function is negatively regulated. However, the role of such phosphorylation in physiological functions, particularly in neuronal functions, is not well understood. Using a phosphorylation-site specific antibody, we detected the phosphorylation of MARCKS at Ser159 by various protein kinases. Rho-kinase, protein kinase A, and protein kinase C, could introduce (32)P into human recombinant MARCKS in vitro and the phosphorylation site was confirmed to be the Ser159 residue. In human neuronal teratoma (NT-2) cells, lysophosphatidic acid (LPA) induced MARCKS phosphorylation dose- and time-dependently. This phosphorylation was sensitive to Rho-kinase inhibitor HA1077. However, the phosphorylation induced by PDBu was lesser sensitive. In a skinned NTera-2 cell system, Ca(2+)-independent and GTP gamma S/ATP-stimulated phosphorylation at Ser159 was also sensitive to pre-treatment C3 toxin and HA1077. These findings suggest that the Ser159 residue of MARCKS is a target of LPA-stimulated Rho-kinase in neuronal cells.
Nagumo
H
H
Frontier 21 Project, Institute for Life Science Research, Asahi Chemical Industry Co., Ltd., 2-1 Samejima, Fuji, Shizuoka 416-8501, Japan.
Ikenoya
M
M
Sakurada
K
K
Furuya
K
K
Ikuhara
T
T
Hiraoka
H
H
Sasaki
Y
Y
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids / Q000494:pharmacology; D008565:Membrane Proteins; D051379:Mice; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009474:Neurons / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D054460:rho-Associated Kinases
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; C000619710:MARCKS protein, human; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015240:Phorbol 12,13-Dibutyrate; D012694:Serine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; C049347:fasudil