Pleckstrin homology domains of phospholipase C-gamma1 directly interact with beta-tubulin for activation of phospholipase C-gamma1 and reciprocal modulation of beta-tubulin function in microtubule assembly.
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.
Chang
Jong-Soo
JS
Department of Life Science, College of Natural Science, Daejin University, Kyeonggido 487-711, Korea. jchang@daejin.ac.kr
Kim
Sung-Kuk
SK
Kwon
Taeg-Kyu
TK
Bae
Sun Sik
SS
Min
Do Sik
DS
Lee
Young Han
YH
Kim
Soon-Ok
SO
Seo
Jeong-Kon
JK
Choi
Jang Hyun
JH
Suh
Pann-Ghill
PG
D000818:Animals; D001798:Blood Proteins; D002460:Cell Line; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D051966:Phospholipase C gamma; D010750:Phosphoproteins; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D051381:Rats; D008941:Spindle Apparatus / Q000737:chemistry; D014404:Tubulin / Q000378:metabolism / Q000502:physiology*; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology
D001798:Blood Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; D014404:Tubulin; C039077:platelet protein P47; D010738:Type C Phospholipases; D051966:Phospholipase C gamma