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J. Biol. Chem., Vol. 280, Issue 8, 6897-6905, February 25, 2005

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Pleckstrin Homology Domains of Phospholipase C-1 Directly Interact with -Tubulin for Activation of Phospholipase C-1 and Reciprocal Modulation of -Tubulin Function in Microtubule Assembly^*

From the ^aDepartment of Life Science, College of Natural Science, Daejin University, Kyeonggido 487-711, Korea, the ^cDepartment of Immunology, College of Medicine, Keimyung University, Taegu 700-712, Korea, the ^dDepartment of Pharmacology, College of Medicine, Pusan National University, Busan 602-739, Korea, the ^eDepartment of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735, Korea, ^fDivision of Molecular and Life Science, College of Science and Technology, Hanyang University, Kyeonggido 426-791, Korea, ^gChem Tech Research Inc., Kyeonggido 472-030, Korea, ^hNano-Digitech Inc., Monmouth Junction, New Jersey 08852, and the ⁱDepartment of Life Science, Pohang University of Science and Technology, Kyungbuk 790-784, Korea

Phosphoinositide-specific phospholipase C-1 (PLC-1) 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-1 PH domain-glutathione S-transferase fusion proteins, followed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry, identified -tubulin as a binding protein of both PLC-1 PH domains. Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of - and -tubulin heterodimers in all eukaryotic cells. PLC-1 and -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-1 was substantially increased in the presence of purified tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that -tubulin activates PLC-1. Furthermore, indirect immunofluorescent microscopy showed that PLC-1 was highly concentrated in mitotic spindle fibers, suggesting that PLC-1 is involved in spindle fiber formation. The effect of PLC-1 in microtubule formation was assessed by overexpression and silencing PLC-1 in COS-7 cells, which resulted in altered microtubule dynamics in vivo. Cells overexpressing PLC-1 showed higher microtubule densities than controls, whereas PLC-1 silencing with small interfering RNAs led to decreased microtubule network densities as compared with control cells. Taken together, our results suggest that PLC-1 and -tubulin transmodulate each other, i.e. that PLC-1 modulates microtubule assembly by -tubulin, and -tubulin promotes PLC-1 activity.

Received for publication, June 8, 2004 , and in revised form, November 9, 2004.

^* This work was supported by Korea Science and Engineering Foundation Grant R05-2001-000-00493-0 and Korea Research Foundation Grant 2002-070-C00074. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^b To whom correspondence should be addressed. Tel.: 82-31-539-1853; Fax: 82-31-539-1850; E-mail: jchang{at}daejin.ac.kr.

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