AKAP200 promotes Notch stability by protecting it from Cbl/lysosome-mediated degradation in Drosophila melanogaster.

AKAP200 is a Drosophila melanogaster member of the "A Kinase Associated Protein" family of scaffolding proteins, known for their role in the spatial and temporal regulation of Protein Kinase A (PKA) in multiple signaling contexts. Here, we demonstrate an unexpected function of AKAP200 in promoting Notch protein stability. In Drosophila, AKAP200 loss-of-function (LOF) mutants show phenotypes that resemble Notch LOF defects, including eye patterning and sensory organ specification defects. Through genetic interactions, we demonstrate that AKAP200 interacts positively with Notch in both the eye and the thorax. We further show that AKAP200 is part of a physical complex with Notch. Biochemical studies reveal that AKAP200 stabilizes endogenous Notch protein, and that it limits ubiquitination of Notch. Specifically, our genetic and biochemical evidence indicates that AKAP200 protects Notch from the E3-ubiquitin ligase Cbl, which targets Notch to the lysosomal pathway. Indeed, we demonstrate that the effect of AKAP200 on Notch levels depends on the lysosome. Interestingly, this function of AKAP200 is fully independent of its role in PKA signaling and independent of its ability to bind PKA. Taken together, our data indicate that AKAP200 is a novel tissue specific posttranslational regulator of Notch, maintaining high Notch protein levels and thus promoting Notch signaling

<i>AKAP200</i> mutants show phenotypes that resemble <i>Notch</i> LOF in adult Drosophila eye and thorax.

<p>(A-C) Tangential adult eye sections with anterior left and dorsal up. (A) Example of <i>wild type</i> (<i>WT)</i> eye tissue identified by presence of pigment granules (shaded grey, also in B and C, in schematics). (B-C) <i>AKAP200</i> mutant tissue, lacking pigment (clonal marker used was <i>w</i>^<i>+</i>, small <i>wt</i> areas shaded grey). (B) Dorsal and (C) ventral eye regions, with <i>AKAP200</i>^<i>M30</i> tissue displaying frequent loss of photoreceptors (schematized in lower panels); see panel (F) for key and quantification. (D-E) Thorax of indicated genotypes, anterior is up. (D) <i>WT</i> control showing normal sensory bristle pattern. (E) <i>AKAP200</i> mutant clones (marked by absence of <i>y</i>^<i>+</i>, some <i>wt</i> patches outlined with yellow line) display defects in SOP specification, resembling Notch signaling defects, as evident by bald spots (white arrow) and supernumerary scutellar bristles (example highlighted by red arrow). (F) Schematic of different ommatidial phenotypes observed in (B-C). Blue box/dots depict loss of one or several outer photoreceptors and green boxes/dots depict loss of R7, with or without simultaneous loss of outer photoreceptors. Graph: quantification of distribution of phenotypes in <i>AKAP200</i>^<i>M30</i> (n = 666 from 8 eyes).</p

AKAP200 effect on Notch depends on lysosomal degradation.

<p>(A-D) Tangential adult eye sections of indicated genotypes and conditions, and (E) quantification of indicated genotypes/conditions; suppression of PR number defects of <i>sev-N</i>^<i>ΔECD</i><i>/+</i> by <i>AKAP200</i> is lost in the presence of 1mg/ml of lysosomal inhibitor, chloroquine (***p<0.0001 from chi square tests, n = 378–644 from 3–4 independent eyes). (F) Quantification (**p = 0.005, *p = 0.02, n = 4) and (G) western blot of third instar larval eye discs showing Notch protein levels in indicated genotypes. Relative to <i>WT</i>, heterozygous <i>AKAP200</i> mutant decreases Notch levels (under control condition, H₂0 treatment), this effect is lost in the presence of 1 mg/ml chloroquine (Y-Tub, bottom, serves as loading control).</p

Association of AKAP200 and Notch.

<p>(A) Notch is co-immunoprecipitated by AKAP200-S: immunoblot from S2 cell whole cell lysates expressing Notch either in combination with Flag-control or AKAP200-S-Flag. Cell lysates were immunoprecipitated with anti-Flag antibody (IP-Flag) and blots were probed with anti-NICD antibody, revealing specific co-IP of Notch with AKAP200-S-Flag with no binding to Flag (right panel-10% input, bottom panel- blots probed with anti-Flag antibody). The specific interaction of AKAP200 and the NICD could be because of the experimental conditions; full length Notch is a large membrane bound protein (270 KDa) it may not be as easily accessible to AKAP200 as the NICD. Given the large size of full length Notch, one cannot exclude the possibility that the physical conformation of the interaction prevents co-immunoprecipitation; for example, AKAP200 maybe buried inside full length Notch. (B) AKAP200-S is coimmunoprecipitated by Notch: immunoblot from S2 cell whole cell lysates expressing AKAP200-S-Flag in combination with GFP-control or Notch-GFP. Cell lysates were immunoprecipitated with anti-GFP antibody (IP-GFP) and blots were probed with anti-Flag antibody, revealing specific co-IP of AKAP200-S-Flag with Notch with no binding to GFP (right panel-10% input, bottom panel- blots probed with anti-GFP antibody). (C) Confocal eye sections from third larval instar eye discs of <i>tub>AKAP200-S-Flag</i> depicting localization AKAP200-S, Notch, and PatJ (marking cellular outlines at junctional level and highlighting developing PR clusters, with strongest staining observed in R2/R5). Note co-localization between anti-NICD punctae and AKAP200-S-Flag (example marked by white arrow), Pearson co-efficient R = 0.67.</p

The effect of AKAP200 on Notch signaling depends on Cbl.

<p><b>(A-D) Tangential adult eye sections of indicated genotypes, with schematics in lower panels (see</b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007153#pgen.1007153.g002" target="_blank">Fig 2A</a><b>for key).</b> (A) PR number defects induced by <i>sev-N</i>^<i>ΔECD</i> are not modified by the heterozygous <i>cbl</i> mutant alone (B), but are suppressed by heterozygous <i>AKAP200</i> mutant (C). (D) Simultaneous reduction of one genomic copy of both <i>cbl</i> and <i>AKAP200</i> reduces the effect of <i>AKAP200</i> suppression of Notch activation. (E) Quantification of genetic interactions of genotypes in (A-D): ***p<0.0001 from chi square tests (n = 320–686 from 3–4 independent eyes). (F-G) Western blot (F) of third instar larval eye disc lysate showing Notch protein expression (blue arrow: full length Notch, black arrow: membrane bound NEXT, red arrow: NICD). Relative to <i>WT</i> (left lane), a reduction in total endogenous Notch protein is observed in <i>AKAP200</i>^<i>M30</i><i>/+</i> (middle lane), which is partially suppressed in lysates from <i>AKAP200</i>^<i>M30</i><i>/+; cbl/+</i> (©-Tub [Y-Tub], bottom, serves as loading control). (G) Quantification of Western blot lanes from (F); n = 3, **p = 0.003, p = 0.01 from student’s t test (error bars represent standard deviations).</p

AKAP200 promotes Notch signaling in a PKA independent manner.

<p>(A) <i>AKAP200</i> has 2 splice variants. AKAP200-L, which can interact with the regulatory subunits of PKA via a tethering site coded for by exon 5 (blue). This exon is spliced out in AKAP200-S, eliminating its ability to interact with PKA. (B-E) Both AKAP200 isoforms can rescue PR number defects in the eye and lethality. Note in schematic of eye phenotypes, loss is indicated by a solid black dot and loss specifically of R7 is indicated by a hollow dot (B) Quantification of genotypes shown in (C-E) ***<i>p</i><0.0001 by chi square test (against <i>AKAP200</i>^<i>M30</i>, n = 514–726, from 3 independent eyes). (C-E) Tangential adult eye sections of indicated genotypes (C) Homozygous <i>AKAP200</i>^<i>M30</i> escaper displays PR number defects. Expression of <i>AKAP200-L</i> (D) and <i>-S</i> (E) via <i>tubulin-Gal4</i> rescues the <i>AKAP200</i> phenotype, suggesting that this phenotype is PKA independent. (F-J) PKA-independent effects of AKAP200 on N signaling. (F) Quantification of genotypes shown in (G-J), ***p<0.0001, by chi square test (n = 320–573 from 3–4 independent eyes). (G-J) Tangential adult eye sections of indicated genotypes. PR number defects caused by <i>sev-N</i>^<i>ΔECD</i> (G) is not modified by <i>PKA</i>^<i>-/+</i> (H), but is suppressed by <i>AKAP200</i> mutant (I), or both together (J). There is no statistical difference in the effect on <i>sev-N</i>^<i>ΔECD</i> of removing either one copy of A<i>KAP200</i> alone or together with <i>PKA</i>, suggesting that PKA may not be required for AKAP200’s effect on Notch signaling.</p