Pleckstrin homology domains of phospholipase C-gamma 1 directly interact with beta-tubulin for activation of phospholipase C-gamma 1 and reciprocal modulation of beta-tubulin function in microtubule assembly
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SCOPUS
- Title
- Pleckstrin homology domains of phospholipase C-gamma 1 directly interact with beta-tubulin for activation of phospholipase C-gamma 1 and reciprocal modulation of beta-tubulin function in microtubule assembly
- Authors
- Chang, JS; Kim, SK; Kwon, TK; Bae, SS; Min, DS; Lee, YH; Kim, SO; Seo, JK; Choi, JH; Suh, PG
- Date Issued
- 2005-02-25
- Publisher
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLO
- Abstract
- Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains, an N-terminal domain and a split PH domain. Here we show that pull down of NIH3T3 cell extracts with PLC-gamma1 PH domain-glutathione S-transferase fusion proteins, followed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry, identified beta-tubulin as a binding protein of both PLC-gamma1 PH domains. Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of alpha- and beta-tubulin heterodimers in all eukaryotic cells. PLC-gamma1 and beta-tubulin colocalized in the perinuclear region in COS-7 cells and cotranslocated to the plasma membrane upon agonist stimulation. Membrane-targeted translocation of depolymerized tubulin by agonist stimulation was also supported by immunoprecipitation analyses. The phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolyzing activity of PLC-gamma1 was substantially increased in the presence of purified tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that beta-tubulin activates PLC-gamma1. Furthermore, indirect immunofluorescent microscopy showed that PLC-gamma1 was highly concentrated in mitotic spindle fibers, suggesting that PLC-gamma1 is involved in spindle fiber formation. The effect of PLC-gamma1 in microtubule formation was assessed by overexpression and silencing PLC-gamma1 in COS-7 cells, which resulted in altered microtubule dynamics in vivo. Cells overexpressing PLC-gamma1 showed higher microtubule densities than controls, whereas PLC-gamma1 silencing with small interfering RNAs led to decreased microtubule network densities as compared with control cells. Taken together, our results suggest that PLC-gamma1 and beta-tubulin transmodulate each other, i.e. that PLC-gamma1 modulates microtubule assembly by beta-tubulin, and beta-tubulin promotes PLC-gamma1 activity.
- Keywords
- PROTEIN-KINASE-C; NUCLEOTIDE EXCHANGE FACTOR; GROWTH-FACTOR RECEPTOR; PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE; POLYMERIZATION DYNAMICS; SIGNALING MOLECULES; ARACHIDONIC-ACID; ALPHA-SUBUNITS; GAMMA-SUBUNITS; PH DOMAINS
- URI
- https://oasis.postech.ac.kr/handle/2014.oak/24752
- DOI
- 10.1074/JBC.M4063502
- ISSN
- 0021-9258
- Article Type
- Article
- Citation
- JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 280, no. 8, page. 6897 - 6905, 2005-02-25
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