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

• Published in: Journal of neurochemistry 2002-04, Vol.81 (1), p.9-16
• Main Authors: Ikenoya, Mami, Hidaka, Hiroyoshi, Hosoya, Takamitsu, Suzuki, Masaaki, Yamamoto, Naoki, Sasaki, Yasuharu
• Format: Article
• Language: English
• Subjects: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine - analogs & derivatives; 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine - chemistry; 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine - pharmacology; Amides - pharmacology; Biological and medical sciences; Cell Line; Dose-Response Relationship, Drug; Enzyme Activation - drug effects; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Fundamental and applied biological sciences. Psychology; Glucosidases; Humans; H‐1152; Indoles - pharmacology; Intracellular Signaling Peptides and Proteins; Isolated neuron and nerve. Neuroglia; lysophosphatidic acid; Lysophospholipids - pharmacology; Membrane Proteins; Myristoylated Alanine-Rich C Kinase Substrate; myristoylated alanine‐rich C kinase substrate (MARCKS); Neurons - cytology; Neurons - drug effects; Neurons - metabolism; NT‐2; Phorbol 12,13-Dibutyrate - pharmacology; Phosphoproteins - metabolism; Phosphorylation - drug effects; Protein Kinase C - antagonists & inhibitors; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Protein-Serine-Threonine Kinases - metabolism; Pyridines - pharmacology; rho GTP-Binding Proteins - metabolism; rho-Associated Kinases; Rho‐kinase; Substrate Specificity; Vertebrates: nervous system and sense organs
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2002.00801.x

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 µm and Y‐27632 at 10–30 µm, 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.