Neuralized-like 1 (Neurl1) targeted to the plasma membrane by N-myristoylation regulates the Notch ligand Jagged1.
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.
Koutelou
Evangelia
E
Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.
Sato
Shigeo
S
Tomomori-Sato
Chieri
C
Florens
Laurence
L
Swanson
Selene K
SK
Washburn
Michael P
MP
Kokkinaki
Maria
M
Conaway
Ronald C
RC
Conaway
Joan W
JW
Moschonas
Nicholas K
NK
D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D015536:Down-Regulation; D004330:Drosophila; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D000072100:Jagged-1 Protein; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D019814:Myristic Acid / Q000378:metabolism*; D000072099:Serrate-Jagged Proteins; D018411:Spodoptera; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D029721:Drosophila Proteins; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; C000606274:Jag1 protein, mouse; D000072100:Jagged-1 Protein; D008024:Ligands; D008565:Membrane Proteins; C000606278:Ser protein, Drosophila; D000072099:Serrate-Jagged Proteins; D025801:Ubiquitin; D019814:Myristic Acid; C556508:NEURL1 protein, human; D044767:Ubiquitin-Protein Ligases; C071006:neur protein, Drosophila