Abstract
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.
MeSH terms
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1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / analogs & derivatives
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1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / chemistry
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1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / pharmacology*
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Amides / pharmacology
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Calcium-Binding Proteins
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Cell Line
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Dose-Response Relationship, Drug
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Enzyme Activation / drug effects
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Enzyme Inhibitors / chemistry
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Enzyme Inhibitors / pharmacology*
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Glucosidases
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Humans
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Indoles / pharmacology
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Intracellular Signaling Peptides and Proteins*
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Lysophospholipids / pharmacology
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Membrane Proteins*
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Myristoylated Alanine-Rich C Kinase Substrate
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Neurons / cytology
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Neurons / drug effects
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Neurons / metabolism*
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Phorbol 12,13-Dibutyrate / pharmacology
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Phosphoproteins / metabolism*
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Phosphorylation / drug effects
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Protein Kinase C / antagonists & inhibitors
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Protein Serine-Threonine Kinases / antagonists & inhibitors*
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Protein Serine-Threonine Kinases / metabolism
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Pyridines / pharmacology
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Substrate Specificity
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rho GTP-Binding Proteins / metabolism
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rho-Associated Kinases
Substances
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2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine
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Amides
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Calcium-Binding Proteins
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Enzyme Inhibitors
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Indoles
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Intracellular Signaling Peptides and Proteins
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Lysophospholipids
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MARCKS protein, human
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Membrane Proteins
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Phosphoproteins
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Pyridines
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Myristoylated Alanine-Rich C Kinase Substrate
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Y 27632
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Phorbol 12,13-Dibutyrate
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1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
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Protein Serine-Threonine Kinases
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rho-Associated Kinases
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Protein Kinase C
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Glucosidases
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PRKCSH protein, human
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rho GTP-Binding Proteins
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Ro 31-8220