Pleckstrin homology domain of phospholipase C-gamma1 directly binds to 68-kDa neurofilament light chain.
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)P(2)] 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.
Kim
Sung Kuk
SK
epartment of Life Science, College of Natural Science, Daejin University, Kyeonggido 487-711, Korea.
Choi
Jang Hyun
JH
Suh
Pann Ghill
PG
Chang
Jong Soo
JS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D026481:Blotting, Far-Western; D005982:Glutathione Transferase / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020932:Nerve Growth Factor / Q000494:pharmacology; D016900:Neurofilament Proteins / Q000737:chemistry / Q000378:metabolism*; D016716:PC12 Cells; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D051966:Phospholipase C gamma / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D025941:Protein Interaction Mapping; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001798:Blood Proteins; D007527:Isoenzymes; D016900:Neurofilament Proteins; D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; C039077:platelet protein P47; D020932:Nerve Growth Factor; D005982:Glutathione Transferase; D051966:Phospholipase C gamma