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

J Neurochem. 2002 Apr;81(1):9-16. doi: 10.1046/j.1471-4159.2002.00801.x.

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

  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / analogs & derivatives
  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / chemistry
  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / pharmacology*
  • Amides / pharmacology
  • Calcium-Binding Proteins
  • Cell Line
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Glucosidases
  • Humans
  • Indoles / pharmacology
  • Intracellular Signaling Peptides and Proteins*
  • Lysophospholipids / pharmacology
  • Membrane Proteins*
  • Myristoylated Alanine-Rich C Kinase Substrate
  • Neurons / cytology
  • Neurons / drug effects
  • Neurons / metabolism*
  • 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
  • Substrate Specificity
  • rho GTP-Binding Proteins / metabolism
  • rho-Associated Kinases

Substances

  • 2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine
  • Amides
  • Calcium-Binding Proteins
  • Enzyme Inhibitors
  • Indoles
  • Intracellular Signaling Peptides and Proteins
  • Lysophospholipids
  • MARCKS protein, human
  • Membrane Proteins
  • Phosphoproteins
  • Pyridines
  • Myristoylated Alanine-Rich C Kinase Substrate
  • Y 27632
  • Phorbol 12,13-Dibutyrate
  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
  • Protein Serine-Threonine Kinases
  • rho-Associated Kinases
  • Protein Kinase C
  • Glucosidases
  • PRKCSH protein, human
  • rho GTP-Binding Proteins
  • Ro 31-8220