Inhibition of rho-kinase-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitor.
The functions of small G protein Rho-associated kinase (Rho-kinase) have been determined in muscle and non-muscle cells, but, particularly in neuronal cells, its effector(s) has not been well known. Recently, we preliminarily reported that Rho-kinase phosphorylates the Ser159 residue in myristoylated alanine-rich C kinase substrate (MARCKS) in vitro, but it remains obscure in vivo. To further clarify this point, we developed an isoquinolinesulfonamide derivative, H-1152, that is a more specific, stronger and membrane-permeable inhibitor of Rho-kinase with a Ki value of 1.6 nM, but poor inhibitor of other serine/threonine kinases. H-1152 dose-dependently inhibited the phosphorylation of MARCKS in human neuroteratoma (NT-2) cells stimulated by Rho-activator lysophosphatidic acid (LPA), which was determined by phosphorylation site-specific antibody against phospho-Ser159 in MARCKS, whereas it hardly inhibited the phosphorylation stimulated by phorbol-12,13-dibutyrate (PDBu). In contrast, two other Rho-kinase inhibitors, HA-1077 at 30 microM and Y-27632 at 10-30 microM, inhibited the phosphorylation of MARCKS in the cells stimulated by LPA and PDBu. A PKC inhibitor Ro-31-8220 selectively inhibited PDBu-induced phosphorylation of MARCKS. Taken together with our previous results, the present findings strongly suggest that Rho/Rho-kinase phosphorylates MARCKS at Ser159 residue in neuronal cells in response to LPA stimulation and that H-1152 is a useful tool to confirm Rho-kinase function(s) in cells and tissues.
Ikenoya
Mami
M
Department of Microbiology and Molecular Virology, School of Medicine, Tokyo Medical and Dental University, Japan.
Hidaka
Hiroyoshi
H
Hosoya
Takamitsu
T
Suzuki
Masaaki
M
Yamamoto
Naoki
N
Sasaki
Yasuharu
Y
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D000577:Amides / Q000494:pharmacology; D002135:Calcium-Binding Proteins; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005959:Glucosidases; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids / Q000494:pharmacology; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D013379:Substrate Specificity; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases
C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine; D000577:Amides; D002135:Calcium-Binding Proteins; D004791:Enzyme Inhibitors; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D011725:Pyridines; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; C108830:Y 27632; D015240:Phorbol 12,13-Dibutyrate; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D005959:Glucosidases; C070368:PRKCSH protein, human; D020741:rho GTP-Binding Proteins; C064758:Ro 31-8220