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J. Biol. Chem., Vol. 281, Issue 38, 27794-27805, September 22, 2006

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The Role of X/Y Linker Region and N-terminal EF-hand Domain in Nuclear Translocation and Ca²⁺ Oscillation-inducing Activities of Phospholipase C, a Mammalian Egg-activating Factor^*

Keiji Kuroda, Masahiko Ito¹, Tomohide Shikano, Takeo Awaji, Ayako Yoda, Hiroyuki Takeuchi, Katsuyuki Kinoshita, and Shunichi Miyazaki

From the Department of Physiology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo 162-8666 and the Department of Obstetrics and Gynecology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8241, Japan

Sperm-specific phospholipase C-zeta (PLC) causes intracellular Ca²⁺ oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLC, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLC mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374–381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg³⁷⁶, Lys³⁷⁷, Arg³⁷⁸, Lys³⁷⁹, or Lys³⁸¹ with glutamate, whereas Ca²⁺ oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys²⁹⁹ and/or Lys³⁰¹ in the C terminus of X domain, or Trp¹³, Phe¹⁴, or Val¹⁸ in the N terminus of EF1. Ca²⁺ oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374–383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296–309 or 1–19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2–4 or C2 domain. Thus, PLC is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLC1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.

Received for publication, April 11, 2006 , and in revised form, July 13, 2006.

^* This work was supported by a grant-in-aid for General Scientific Research (B) (to S. M.) from the Japan Ministry of Education, Science, Sports, and Culture. 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.

¹ To whom correspondence should be addressed: Dept. of Physiology, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel.: 81-3-5269-7414; Fax: 81-3-5269-7414; E-mail: mito{at}research.twmu.ac.jp.

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