Exp Mol Med.  2006 Jun;38(3):265-272.

Pleckstrin homology domain of phospholipase C-gamma1 directly binds to 68-kDa neurofilament light chain

• Kim SK
• Choi JH
• Suh PG
• Chang JS

Abstract

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.

Keyword

neurofilament protein L; PC12 cells; phospholipase C gamma; phosphatidylinositol 4,5-diphosphate; protein interaction mapping

MeSH Terms

Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Rats
Protein Interaction Mapping
Protein Biosynthesis/drug effects
Protein Binding/drug effects
Phosphoproteins/chemistry/*metabolism
Phospholipase C gamma/antagonists & inhibitors/chemistry/*metabolism
Phosphatidylinositol 4,5-Diphosphate/metabolism
Peptides/chemistry/metabolism
PC12 Cells
Neurofilament Proteins/chemistry/*metabolism
Nerve Growth Factor/pharmacology
Molecular Weight
Molecular Sequence Data
Microtubules/metabolism
Microscopy, Fluorescence
Isoenzymes/metabolism/pharmacology/physiology
Glutathione Transferase/metabolism
Blotting, Far-Western
Blood Proteins/chemistry/*metabolism
Binding Sites
Animals
Amino Acid Sequence