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 (PIP(2)) 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.
Highlights
PLC-␥11 is an important signaling molecule for cell proliferation and differentiation
Our present study demonstrates that the pleckstrin homology (PH) domains of PLC-␥1 interact with -tubulin in vivo
The PH domains of PLC-␥1 have been shown to facilitate membrane targeting via interactions with phosphoinositide [7] and activate enzymatic activity via protein-protein interactions [26]
Summary
Antibodies—Monoclonal anti--tubulin and anti-␣-tubulin were purchased from Chemicon (Temecula, CA) and Sigma, and polyclonal anti-tubulin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The membranes were washed in TBT buffer, and bound proteins were detected by incubation with a secondary HRP-conjugated anti-goat antibody and visualized with an ECL detection system. The pellet was resuspended in ice-cold tubulin polymerizing buffer, homogenized, incubated on ice for depolymerization, and centrifuged at 100,000 ϫ g These polymerization/depolymerization cycles were repeated two times, and the supernatants and pellets from each step were resolved by 10% SDS-PAGE and transferred to PVDF membranes for Western immunoblotting using an anti-PLC-␥1 antibody. The cells were fixed at 37 °C for 10 min in 4% paraformaldehyde and incubated with affinity-purified monoclonal anti-FLAG or polyclonal anti-PLC-␥1 antibodies for 1 h at room temperature in a humidity chamber. B, the bound proteins were resolved by 10% SDS-PAGE followed by Western blotting with a monoclonal anti--tubulin antibody (mAb TU-20). The effect of -tubulin was examined by adding the indicated amounts of ␣- and -tubulin to the PLC-␥1 assay mixture
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