Casein kinase 1 family proteins promote Slimb-dependent Expanded degradation
- Queen Mary University of London, United Kingdom
- Washington University School of Medicine, United States
- The Francis Crick Institute, United Kingdom
Figures
Figure 1 with 1 supplement
Gish, the Drosophila orthologue of CkIγ interacts with Ex in a Crb-dependent manner.
(A) An AP-MS approach identified Gish as an Ex-interacting protein. Summary table with AP-MS results for Gish and Hpo. CG# denotes Flybase CG number, while unique and total denote the number of …
Figure 1—figure supplement 1
Evolutionary conservation of CKIs and features of Gish.
(A) Phylogenetic relation between human and Drosophila CKI isoforms and other Drosophila kinases with >50% overall similarity to CkIα. (B) Domain structure of isoforms F and I of Gish. Red rectangle …
Figure 2 with 1 supplement
CKI kinases promote Ex phosphorylation and depletion.
(A) and (B) Gish promotes Ex phosphorylation and depletion in a kinase- and sub-cellular localisation-dependent manner. (A) S2 cells were used to co-transfect V5-tagged Ex1-468 with GFP and Crbintra,…
Figure 2—figure supplement 1
Molecular requirements for the effect of CKIs in the regulation of Ex protein stability.
(A) Expression of CKIs promotes depletion of Ex protein levels. Graph depicts results of quantification of Ex1-468 protein levels assessed by Western blotting analysis. Ex protein levels were …
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Figure 2—figure supplement 1—source data 1
Source data for quantification of Expanded protein levels from Western blot analyses performed in Drosophila S2 cells.
- https://doi.org/10.7554/eLife.46592.006
Figure 3 with 1 supplement
Crb-, Slmb- and CKI-mediated regulation of an in vivo Ex protein stability reporter.
(A–C) Crb and Slmb regulate localisation and in vivo levels of the ubi-Ex1-468::GFP reporter. Confocal micrographs of transverse sections of wing imaginal discs from wandering third instar larvae …
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Figure 3—source data 1
Source data for quantification of relative protein levels of Ex::GFP in vivo reporter.
- https://doi.org/10.7554/eLife.46592.010
Figure 3—figure supplement 1
Characterisation of the Ex stability reporter and in vivo regulation of Ex stability and tissue growth.
(A) and (B) Crb and Slmb regulate localisation and levels of the ubi-Ex1-468::GFP reporter. XY confocal micrograph of wing imaginal discs from wandering third instar larvae carrying clones mutant …
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Figure 3—figure supplement 1—source data 1
Source data for quantification of adult wing sizes and relative protein levels of Crb and Ex S453A::GFP reporter.
- https://doi.org/10.7554/eLife.46592.009
Figure 4 with 1 supplement
CkIα and Gish are required for Crb-induced Ex degradation.
(A) and (B) RNAi-mediated depletion of gish abrogates Crb-induced Ex degradation. FLAG-tagged Ex1-468 (A) or V5-tagged ExFL (B) were co-expressed with Crbintra or CrbΔFBM, in the presence of dsRNA …
Figure 4—figure supplement 1
Role of CKI kinases and alternative kinases in the regulation of Ex stability.
(A) Validation of gish RNAi tools. S2 cells were treated with the indicated dsRNA 24 hr before transfection with the indicated constructs. Expression of FLAG-tagged Gish and RNAi efficiency were …
Figure 5 with 2 supplements
Loss of function of ckIα, but not gish or dco, modulates levels of an Ex protein stability reporter in vivo.
(A) dco mutant clones do not affect the Ex in vivo stability reporter. XY (A, A’) and transverse sections (A’’, A’’’) of ubi-Ex1-468::GFP-expressing third instar wing imaginal discs containing dcole8…
Figure 5—figure supplement 1
In vivo clonal analysis of the role of CKI kinases in the regulation of the Ex stability reporter.
(A) gishKG03891 mutant clones do not affect Ex1-468::GFP levels. XY (A and A’) and transverse (A’’ and A’’’) confocal micrographs of third instar wing discs carrying gishKG03891 mutant clones …
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Figure 5—figure supplement 1—source data 1
Source data for quantification of aPKC protein levels in wing imaginal discs carrying clones mutant forckIα.
- https://doi.org/10.7554/eLife.46592.016
Figure 5—figure supplement 2
Role of Slmb and CkIα in regulating an in vivo Ex protein stability reporter in eye, leg and haltere discs.
(A–F) Slmb and CkIα regulate levels of the ubi-Ex1-468::GFP reporter in leg and haltere discs, but not eye discs. Confocal micrographs of transverse sections of eye (posterior to the morphogenetic …
Figure 6 with 1 supplement
RNAi-mediated depletion of ckIα, but not gish, suppresses Crbintra-induced degradation of an in vivo Ex protein stability reporter.
(A) and (B) ckIα knockdown has a minimal effect on Ex1-468::GFP levels.(C) and (D) ckIα RNAi blocks Crbintra-mediated depletion of Ex1-468::GFP. (E–H) gish RNAi-mediated depletion does not …
Figure 6—figure supplement 1
Analysis of the effect of RNAi-mediated depletion and clonal mutation of CKI kinases in the regulation of Crbintra-induced Ex degradation.
(A) Temporally-controlled expression of Crbintra promotes Ex1-468::GFP degradation in vivo. XY (A, A’, Aa and Aa’) and transverse sections (A’’, A’’’, Aa’’ and Aa’’’) of third instar wing imaginal …
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Figure 6—figure supplement 1—source data 1
Source data for quantification of relative levels of the Ex::GFPin vivoreporter in wing imaginal discs expressing RNAi targeting CKI kinases alone or in combination with expression of Crbintra.
- https://doi.org/10.7554/eLife.46592.019
Figure 7
Effect of CkIα and Gish loss- and gain-of-function on Yki target gene expression.
(A–D) Yki transcriptional activity is reduced by loss of ckIα, but unaffected by loss of gish. XY and XZ confocal images of third instar wing imaginal discs bearing clones mutant for ckIα8B12 (A, C, …
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Figure 7—source data 1
Source data for quantification of relative levels of the Ex::GFPin vivoreporter in wing imaginal discs expressing RNAi targeting CKI kinases alone or in combination with expression of Crbintra.
- https://doi.org/10.7554/eLife.46592.021
Author response image 1
Overexpression of CkIα, but not GishisoI, causes a mild overgrowth of the posterior compartment of the wing.
(A-C) Adult wings from female flies overexpressing no transgene (A), UAS-CkIα (B), or UAS-GishisoI (C), in the posterior compartment of the developing wing under the control of hhGal4. Crosses were …
Author response image 2
Evaluation of rabbit Ex phospho-S453 antibody in Drosophila S2 cells.
S2 cells were transfected with the indicated plasmids 48h prior to lysis and processing for Western blot analysis. Note that the phospho-S453 Ex antibody does not recognise any protein in the …
Author response image 3
Evaluation of sheep Ex phospho-S453 antibody in Drosophila S2 cells.
S2 cells were transfected with the indicated plasmids 48h prior to lysis and processing for immunoblotting. Note that the phospho-S453 Ex antibody does not seem to specifically recognise full length …
Author response image 4
Evaluation of Ex antibody in the recognition of endogenous Ex protein.
S2 cells were transfected with the indicated plasmids 48h prior to lysis and dsRNA treatment was performed 24h before transfection. Note that there are several bands recognised by the Ex antibody. …
Author response image 5
Evaluation of Ex electrophoretic mobility shift using Phos-tag gels.
S2 cells were transfected with the indicated plasmids 48h prior to lysis. Lysates were run in Phos-tag gels to assess phosphorylation pattern of Ex1-468 (WT, S453A, S457A or S462A) in the presence …
Author response image 6
ckIαRNAi partially reverts Crbintra-induced upregulation of Yki target genes and overgrowth.
(A-E) ckIαRNAi partially reverts Crbintra-induced upregulation of the Yki activity reporter ex-lacZ. Sum slices projections of z-stacks of the pouch region of third instar wing imaginal discs …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.46592.022
