Evidence that the C‐terminal domain (CtD) autoinhibits neural repression by <i>Drosophila</i> E(spl)M8

Kahali, Bhaskar; Kim, Jee‐Eun; Karandikar, Umesh C.; Bishop, Clifton P.; Bidwai, Ashok P.

doi:10.1002/dvg.20581

Cited by 16 publications

(29 citation statements)

References 59 publications

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“…As previously described by others and us, full-length M8 and the isolated CtD interact robustly with Gro (Fig. 2, and see [28, 29, 35, 42, 43]). However, placement of the WRPW motif immediately C-terminal to the CK2 site, S 159 DCD (see Fig.…”

Section: Resultssupporting

confidence: 73%

“…The absence of this inhibitory domain in M8* likely mediates the dominant eye defects of E(spl)D . Further support to this model was the finding that the isolated CtD could impair Notch signaling in vivo, and negate the dominant activity of the non-inhibited proteins M8* and M8-S 159 D [35]. Together, these studies implicated inter-domain interactions in repression by M8.…”

Section: Introductionmentioning

confidence: 94%

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New insights into the Orange domain of E(spl)-M8, and the roles of the C-terminal domain in autoinhibition and Groucho recruitment

et al. 2011

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CK2 is a Ser/Thr protein kinase that regulates the activity of the Drosophila basic-helix-loop-helix (bHLH) repressor M8 encoded by the Enhancer of split Complex (E(spl)C) during neurogenesis. Specifically, phosphorylation appears to elicit a conformational change in an autoinhibited state of M8 to one that is permissive for repression. We describe biochemical and molecular modeling studies that provide new insights into repression by M8. Our studies implicate the phosphorylation domain in autoinhibition, and indicate that binding of the co-repressor Groucho (Gro) is context-dependent. Molecular modeling indicates that the Orange domain, proposed to be a specificity-determinant, may instead play a structural role, and that a conformational rearrangement of this domain may be necessary for repression. This model also provides a structural mechanism for the behavior of mutant alleles of the m8 gene. The insights gained from these studies should be applicable to the conserved metazoan bHLH repressors of the Hairy and Enhancer of Split (HES) family that are related to Drosophila M8.

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Section: Resultssupporting

confidence: 73%

Section: Introductionmentioning

confidence: 94%

New insights into the Orange domain of E(spl)-M8, and the roles of the C-terminal domain in autoinhibition and Groucho recruitment

et al. 2011

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“…Computational analysis of fly E(spl) proteins reveals that the P-domain and its flanking sequences (aside from WRPW) are intrinsically disordered (ID), as is also the case for the HES/HER proteins (ref [64], and data not shown). It is becoming increasingly apparent that ID-regions, which are rich in Ser/Thr and Asp/Glu residues, are used to regulate activity through PTM [77]–[79].…”

Section: Discussionmentioning

confidence: 89%

“…2), an approach that has been used to compare eye size [43], [48], [63], [64]. The facet number for N spl males, 320±10, was used as a baseline.…”

Section: Resultsmentioning

confidence: 99%

The Ser/Thr Phosphatase PP2A Regulatory Subunit Widerborst Inhibits Notch Signaling

2014

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Drosophila Enhancer of split M8, an effector of Notch signaling, is regulated by protein kinase CK2. The phosphatase PP2A is thought to play an opposing (inhibitory) role, but the identity of the regulatory subunit was unknown. The studies described here reveal a role for the PP2A regulatory subunit widerborst (wdb) in three developmental contexts; the bristle, wing and the R8 photoreceptors of the eye. wdb overexpression elicits bristle and wing defects akin to reduced Notch signaling, whereas hypomorphic mutations in this PP2A subunit elicit opposite effects. We have also evaluated wdb functions using mutations in Notch and E(spl) that affect the eye. We find that the eye and R8 defects of the well-known Nspl mutation are enhanced by a hypomorphic allele of wdb, whereas they are strongly rescued by wdb overexpression. Similarly, ectopic wdb rescues the eye and R8 defects of the E(spl)D mutation, which affects the m8 gene. In addition, wdb overexpression also rescues the bristle defects of ectopically expressed M8, or the eye and R8 defects of its CK2 phosphomimetic variant M8-S159D. The latter finding suggests that PP2A may target M8 at highly conserved residues in the vicinity of the CK2 site, whose phosphorylation controls repression of Atonal and the R8 fate. Together, the studies identify PP2A-Wdb as a participant in Notch signaling, and suggest that M8 activity is controlled by phosphorylation and dephosphorylation. The conservation of the phosphorylation sites between Drosophila E(spl) and the HES/HER proteins from mammals, reptiles, amphibians, birds and fish raises the prospect that this mode of regulation is widespread.

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“…To date, only E(spl)M8 (referred to as M8) has emerged as an apparent genetic link between N and EGFR-MAPK (Bandyopadhyay et al, 2016, Majot et al, 2015). Both genetic and biochemical evidence suggest that M8 directly antagonizes Ato via its 5’ enhancer following the formation of IGs (Majot and Bidwai, 2017, Karandikar et al, 2004, Kahali, et al, 2010). N and Su(H) are required for the expression of M8 and genetic evidence suggests that MAPK phosphorylates the C-terminal domain of M8 to potentiate M8’s repression of Ato (Bandyopadhyay et al, 2016; Bose et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

DrosophilaAop imposes a delay on E(spl)-mediated repression of Ato during R8 specification

et al. 2019

Preprint

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22Drosophila retinal patterning requires the expression of Atonal (Ato) through coordinated 23 regulation of 5' and 3' enhancer modules. ato-3' directs initial expression of Ato which then 24 directs autoregulation via 5'-ato. Notch (N) signaling also regulates 5'-ato, first enhancing Ato 25 expression and later repressing Ato by inducing E(spl) bHLHs. N signaling balances these 26 opposing functions by directing its obligate nuclear transcription factor, Suppressor of Hairless 27 (Su(H)), only in repressing 5'-ato. In this study, we reveal a novel and more nuanced role for 28 Su(H) in its regulation of 5'-ato. During retinal patterning, Su(H) is required for the expression 29 Anterior open (Aop), which, in turn, promotes 5'-ato activity. We demonstrate that Aop is 30 induced early in retinal patterning via N pathway activity, wherein Aop is required cell-31 autonomously for robust Ato expression during photoreceptor specification. In aop mutants, 32 expression from both ato enhancers is perturbed, suggesting that Aop promotes the Ato 33 autoregulation through maintenance of ato-3' activity. Clonal analysis indicates that Aop 34 indirectly opposes E(spl)-mediated repression of Ato. In the absence of both Aop and E(spl), 35 Ato expression is restored and the founding ommatidial photoreceptors, R8s, are specified. 36 These findings suggest that N signaling, through a potentially conserved relationship with Aop, 37 imposes a delay on ato repression, thus permitting autoregulation and retinogenesis. 38 3 39 Author Summary: 40The eye of the fruit fly has served as a paradigm to understand tissue patterning.41 Complex intercellular signaling networks cooperate during retinal development to allow cells to 42 become specialized visual-system precursor neurons at a specific time and place. These 43 neurons are precisely spaced within the developing retina and later recruit other cells to form 44 the repeated units that comprise insect eyes. The exact placement of each precursor cell 45 precipitates from the precise regulation of the atonal gene, which is first expressed in a cluster 46 of (10-20) cells before becoming restricted to only one cell from each cluster. The Notch 47 signaling pathway is required for both aspects of atonal regulation, first permitting up-regulation 48 within each cluster, and then the subsequent down-regulation to a single cell. However, the 49 connection between these two modes of Notch signaling had remained unclear. In this report, 50 we have identified that the anterior open gene is required to impose a delay on the restrictive 51 mode of Notch signaling, permitting the initial up-regulation of atonal to occur freely. In flies 52 mutant for anterior open, atonal bypasses its own up-regulation and proceeds directly to its 53 singled-out pattern but with significantly diminished robustness than occurs normally.

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Evidence that the C‐terminal domain (CtD) autoinhibits neural repression by Drosophila E(spl)M8

Cited by 16 publications

References 59 publications

New insights into the Orange domain of E(spl)-M8, and the roles of the C-terminal domain in autoinhibition and Groucho recruitment

New insights into the Orange domain of E(spl)-M8, and the roles of the C-terminal domain in autoinhibition and Groucho recruitment

The Ser/Thr Phosphatase PP2A Regulatory Subunit Widerborst Inhibits Notch Signaling

DrosophilaAop imposes a delay on E(spl)-mediated repression of Ato during R8 specification

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