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J. Biol. Chem., Vol. 283, Issue 7, 3846-3853, February 15, 2008

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Neuralized-like 1 (Neurl1) Targeted to the Plasma Membrane by N-Myristoylation Regulates the Notch Ligand Jagged1^*

Evangelia Koutelou¹, Shigeo Sato, Chieri Tomomori-Sato, Laurence Florens, Selene K. Swanson, Michael P. Washburn, Maria Kokkinaki^¶2, Ronald C. Conaway^||, Joan W. Conaway^||3, and Nicholas K. Moschonas^¶4

From the Stowers Institute for Medical Research, Kansas City, Missouri 64110, the Joint Graduate Program in Molecular Biology and Biomedicine, Department of Biology, University of Crete, Heraklion, 71409, Crete, Greece, the ^¶Department of Biology, University of Crete, Heraklion, 71409, Crete, Greece, and the ^||Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160

Notch signaling constitutes an evolutionarily conserved mechanism that mediates cell-cell interactions in various developmental processes. Numerous regulatory proteins interact with the Notch receptor and its ligands and control signaling at multiple levels. Ubiquitination and endocytosis followed by endosomal sorting of both the receptor and its ligands is essential for Notch-mediated signaling. The E3 ubiquitin ligases, Neuralized (Neur) and Mind Bomb (Mib1), are crucial for regulating the activity and stability of Notch ligands in Drosophila; however, biochemical evidence that the Notch ligands are directly targeted for ubiquitination by Neur and/or Mib1 has been lacking. In this report, we explore the function of Neurl1, a mouse ortholog of Drosophila Neur. We show that Neurl1 can function as an E3 ubiquitin ligase to activate monoubiquitination in vitro of Jagged1, but not other mammalian Notch ligands. Neurl1 expression decreases Jagged1 levels in cells and blocks signaling from Jagged1-expressing cells to neighboring Notch-expressing cells. We demonstrate that Neurl1 is myristoylated at its N terminus, and that myristoylation of Neurl1 targets it to the plasma membrane. Point mutations abolishing either Neurl1 myristoylation and plasma membrane localization or Neurl1 ubiquitin ligase activity impair its ability to down-regulate Jagged1 expression and to block signaling. Taken together, our results argue that Neurl1 at the plasma membrane can affect the signaling activity of Jagged1 by directly enhancing its ubiquitination and subsequent turnover.

Received for publication, August 20, 2007 , and in revised form, November 26, 2007.

^* This work was supported by the Stowers Institute for Medical Research and by a Research and Education Action Program PYTHAGORAS II no. 2077 of the Greek Ministry of Education and the European Union (25% from national funds and 75% from the European Social Fund) (to N. K. M). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1–S4, Figs. S1–S5, and other materials.

¹ Recipient of a predoctoral fellowship from the Propondis Foundation.

² Present address: Dept. of Biochemistry and Cell Biology, Georgetown University, Washington D. C. 20057.

³ To whom correspondence may be addressed: Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110. Tel.: 816-926-4091; Fax: 816-926-2091; E-mail: jlc{at}stowers-institute.org. ⁴ To whom correspondence may be addressed: Laboratory of Biology, School of Medicine, University of Patras, University Campus 26500, Patras, Greece. Tel.: 302610997689; Fax: 302610991769; E-mail: n_moschonas{at}med.upatras.gr.

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