Involvement of a proline-rich motif and RING-H2 finger of Deltex in the regulation of Notch signaling

Abstract

The Notch pathway is an evolutionarily conserved signaling mechanism that is essential for cell-cell interactions. The Drosophila deltex gene regulates Notch signaling in a positive manner, and its gene product physically interacts with the intracellular domain of Notch through its N-terminal domain. Deltex has two other domains that are presumably involved in protein-protein interactions: a proline-rich motif that binds to SH3-domains, and a RING-H2 finger motif. Using an overexpression assay, we have analyzed the functional involvement of these Deltex domains in Notch signaling. The N-terminal domain of Deltex that binds to the CDC10/Ankyrin repeats of the Notch intracellular domain was indispensable for the function of Deltex. A mutant form of Deltex that lacked the proline-rich motif behaved as a dominant-negative form. This dominant-negative Deltex inhibited Notch signaling upstream of an activated, nuclear form of Notch and downstream of full-length Notch, suggesting the dominant-negative Deltex might prevent the activation of the Notch receptor. We found that Deltex formed a homo-multimer, and mutations in the RING-H2 finger domain abolished this oligomerization. The same mutations in the RING-H2 finger motif of Deltex disrupted the function of Deltex in vivo. However, when the same mutant was fused to a heterologous dimerization domain (Glutathione-S-Transferase), the chimeric protein had normal Deltex activity. Therefore, oligomerization mediated by the RING-H2 finger motif is an integral step in the signaling function of Deltex.

Functions and Molecular Mechanisms of Deltex Family Ubiquitin E3 Ligases in Development and Disease

“…Domain II of Dx contains a proline-rich motif, which is the binding site of the SH3 domain, that primarily regulates the interaction with other proteins, such as growth factor receptor-binding protein 2 (Grb2) (Matsuno et al, 1998). Lacking the proline-rich motif negatively reverses the Dx regulation of Notch signaling pathway (Matsuno et al, 2002). The C-terminal structures of Drosophila Dx, MDTXs, and human DTXs are highly evolutionarily conserved according to their amino acid sequence and crystal structure alignment (Kishi et al, 2001;Takeyama et al, 2003;Chatrin et al, 2020).…”

“…The C-terminus of Dx contains a RING-H2 domain with E3 ligase activity (Takeyama et al, 2003). In the integral steps of Dx regulated signaling pathway, the formation of homo-multimeric Dx is mediated by the RING-H2 domain (Matsuno et al, 2002). The RING-H2 domain of the DTX family adopts a novel circular fold with eight conserved cysteine and histidine residues, which is different from other RINGs (Miyamoto et al, 2019), whereas DTX3 and DTX3L contain a RING-HC structure with a single histidine (Takeyama et al, 2003).…”

E3 Ubiquitin Ligase Regulators of Notch Receptor Endocytosis: From Flies to Humans

“…These WWE motifs are responsible for binding to the ankyrin repeats of Notch [ 21 ]. Domain II consists of a proline-rich SH3-binding motif (PPLP), whilst the most conserved region of Dx, Domain III contains a RING-H2 zinc-finger domain [ 30 ] followed by a Deltex carboxy-terminal (DTC) domain [ 31 ]. Though there is only one Deltex protein in Drosophila , there are five known Deltex paralogues in humans (referred to as DTX 1, 2, 3, 3L and 4) which are all characterised by the RING-H2 zinc-finger domain in the C-terminus.…”

“…All Deltex paralogues except for DTX3 and DTX3L have two tandem WWE motifs [ 33 ]. DTX1, DTX3 and DTX4 contain a PPLP, SH3-binding motif that, in Drosophila , has been shown to bind to GRB2 (growth factor receptor binding protein 2), an adaptor protein that positively regulates Ras signalling [ 30 , 33 , 34 ]. All five human DTX proteins contain the RING-H2 zinc-finger domain followed by the DTC domain [ 31 ].…”

The Ubiquitin Conjugating Enzyme UbcD1 is Required for Notch Signaling Activation During Drosophila Wing Development

“…In the signal receiving cells, Notch molecules are ubiquitinated by E3 ligases Nedd4 and Suppressor of deltex [Su(dx)] and targeted for lysosomal degradation to avoid ligand independent activation ( Cornell et al, 1999 ; Mazaleyrat et al, 2003 ; Sakata et al, 2004 ; Wilkin et al, 2004 ; Dalton et al, 2011 ). The E3 ubiquitin ligase Deltex (Dx) was isolated as a positive regulator of Notch signaling which genetically and physically interacts with Notch ( Xu and Artavanis-Tsakonas, 1990 ; Diederich et al, 1994 ; Matsuno et al, 1995 ; Matsuno et al, 2002 ). Subsequent studies reveal that Dx promotes ubiquitination and ligand independent activation of Notch through the endocytic machinery ( Hori et al, 2004 ; Wilkin et al, 2008 ; Hori et al, 2011 ; Yamada et al, 2011 ).…”