Pleckstrin homology domains of phospholipase C-γ1 directly interact with β-tubulin for activation of phospholipase C-γ1 and reciprocal modulation of β-tubulin function in microtubule assembly

Jong Soo Chang, Sung Kuk Kim, Taeg Kyu Kwon, Sun Sik Bae, Do Sik Min, Young Han Lee, Soon Ok Kim, Jeong Kon Seo, Jang Hyun Choi, Pann Ghill Suh

Research output: Contribution to journalArticlepeer-review

34 Citations (Scopus)

Abstract

Phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) 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-γ1 PH domain-glutathione S-transferase fusion proteins, followed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry, identified β-tubulin as a binding protein of both PLC-γ1 PH domains. Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of α- and β-tubulin heterodimers in all eukaryotic cells. PLC-γ1 and β-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-γ1 was substantially increased in the presence of purified tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that β-tubulin activates PLC-γ1. Furthermore, indirect immunofluorescent microscopy showed that PLC-γ1 was highly concentrated in mitotic spindle fibers, suggesting that PLC-γ1 is involved in spindle fiber formation. The effect of PLC-γ1 in microtubule formation was assessed by overexpression and silencing PLC-γ1 in COS-7 cells, which resulted in altered microtubule dynamics in vivo. Cells overexpressing PLC-γ1 showed higher microtubule densities than controls, whereas PLC-γ1 silencing with small interfering RNAs led to decreased microtubule network densities as compared with control cells. Taken together, our results suggest that PLC-γ1 and β-tubulin transmodulate each other, i.e. that PLC-γ1 modulates microtubule assembly by β-tubulin, and β-tubulin promotes PLC-γ1 activity.

Original languageEnglish
Pages (from-to)6897-6905
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number8
DOIs
Publication statusPublished - 2005 Feb 25

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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