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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nat Cell Biol. Author manuscript; available in PMC Oct 1, 2012.
Published in final edited form as:
PMCID: PMC3319494
NIHMSID: NIHMS352863
Cdk5 and Mekk1 mediate a Pro-Apoptotic Signaling Response to Endoplasmic Reticulum Stress in a Drosophila Model of Autosomal Dominant Retinitis Pigmentosa
Min-Ji Kang, Jaehoon Chung, and Hyung Don Ryoo
Department of Cell Biology, New York University School of Medicine 550 First Avenue, New York, NY 10016
Corresponding Author: Hyung Don Ryoo Tel: 212-263-7257 ; Hyungdon.Ryoo/at/nyumc.org
Chronic stress in the endoplasmic reticulum (ER) underlies many degenerative and metabolic diseases involving apoptosis of vital cells. A well-established example is Autosomal Dominant Retinitis Pigmentosa (ADRP), an age-related retinal degenerative disease caused by mutant rhodopsins 1, 2. Similar mutant alleles of Drosophila rhodopsin-1 also impose stress on the ER and cause age-related retinal degeneration in that organism 3. Well-characterized signaling responses to ER-stress, referred to as the Unfolded Protein Response (UPR) 4, induce various ER quality control genes that can suppress such retinal degeneration 5. However, how cells activate cell death programs after chronic ER-stress remains poorly understood. Here, we report the identification of a signaling pathway mediated by cdk5 and mekk1 required for ER-stress-induced apoptosis. Inactivation of these genes specifically suppressed apoptosis, without affecting other protective branches of the UPR. Cdk5 phosphorylates Mekk1, and together, activate the JNK pathway for apoptosis. Moreover, disruption of this pathway can delay the course of age-related retinal degeneration in a Drosophila model of ADRP. These findings establish a previously unrecognized branch of ER-stress response signaling involved in degenerative diseases.
Three branches of the Unfolded Protein Response (UPR) are particularly well characterized in mammals and conserved in Drosophila 4. In brief, these pathways involve transmembrane proteins ATF6, IRE1 and PERK, respectively, that can sense stress in the ER lumen. ATF6 is a transcription factor anchored to the ER membrane that translocates to the nucleus after ER-stress triggers its proteolysis, and IRE1 is an endonuclease that activates the transcription factor XBP1 through an unconventional mRNA splicing mechanism. PERK is an ER-stress responsive kinase that mediates the translational activation of the transcription factor, ATF4. The predominant effect of these pathways is to reduce stress in the ER and help the cells return to their normal physiological state. Consistently, the major targets of these transcription factors include genes that encode ER chaperones, anti-oxidant proteins and those involved in misfolded protein degradation 68.
Our in vivo model for ER-stress-induced apoptosis is based on a mutant Drosophila Rhodopsin-1 (Rh-1) allele, Rh-1G69D, which is similar in nature with human rhodopsin mutants that underlie retinal degeneration in Autosomal Dominant Retinitis Pigmentosa (ADRP) 9, 10. While the endogenous allele causes late-onset retinal degeneration without affecting the external eye morphology, overexpression of this encoded protein in larval eye imaginal discs (during photoreceptor differentiation) led to an easily identifiable adult eye phenotype by eclosion (Figure 1A, B, Supplementary Figure S1B). The adult eye was abnormally small, indicative of massive cell loss, and the surviving eye tissue showed a glassy surface that was devoid of ommatidial structures. The effect of Rh-1G69D overexpression can be attributed to excessive ER-stress for the following reasons: The Rh-1G69D overexpression phenotype was suppressed by the co-expression of Drosophila hrd1 (Supplementary Figure S1C), which encodes an E3 ubiquitin ligase dedicated to degrading misfolded ER proteins 5. In addition, we detected signs of ER stress using two independent reporters. One is the XBP1-EGFP reporter, which expresses EGFP in frame only when ER-stress stimulates IRE1-dependent XBP1 mRNA splicing 3. This reporter was activated in Rh-1G69D misexpressing imaginal discs while not active in control tissues (Supplementary Figure S1D, E). We were also able to detect signs of ER-stress through an antibody against Drosophila ATF4. This protein is encoded in the cryptocephal (crc) locus 11. As in mammals 12, we found that the Drosophila ATF4 expression was induced after ER stress (Supplementary Figure S1F, G, H). Expression of Rh-1G69D in eye imaginal discs also increased the level of endogenous superoxides as evidenced by Dihydroethidium (DHE) labeling (Supplementary Figure S1J, K), consistent with previous reports of elevated ROS in stressed ER 1317. Co-expressing Hrd1 suppressed such induction of ATF4 and ROS (Supplementary Figure S1I, L), indicating that these markers appear as a result of misfolded protein overload in the ER.
Figure 1
Figure 1
Cdk5 and its regulatory subunit p35 (Cdk5alpha) are required for Rh-1G69D-induced apoptosis
An easily detectable adult eye phenotype allowed us to conduct an in vivo RNAi screen to identify genes required for Rh-1G69D-induced toxicity. We specifically focused on kinases and phosphatases that could serve as signaling proteins potentially linking the distressed ER and the apoptotic machinery. Of the196 protein kinases and 66 protein phosphatases encoded in the Drosophila genome 18, we were able to target 119 kinases and 39 phosphatases through RNAi mediated knock down, using a total of 276 inverted repeat transgenes available from the Vienna Drosophila RNAi Center (Supplementary Information, Table 1). We found three lines that strongly suppressed the adult eye phenotype, two of which (VDRC35855 and VDRC35856) targeted Drosophila cdk5 (Figure 1C). Cdk5 is an atypical cyclin-dependent kinase with established roles in differentiated postmitotic cells, such as neurons, adipose tissue and pancreatic beta-islet cells 1922. In mammals, Cdk5 is reportedly activated by various stress conditions, including those that disrupt ER function 23. Excessive activation of Cdk5 contributes to neurotoxicity in Alzheimer's and Parkinson's Diseases models 24, 25. We found that cdk5 knockdown did not affect an independent cell death phenotype caused by p53-overexpression in the eye (Figure 1E, F). These results indicate that cdk5 mediates a specific signaling response to mutant Rh-1, rather than affecting the general cell death machinery. When eye imaginal discs were inspected, we noticed a dramatic reduction of TUNEL positive cells, indicating that cdk5 is required for apoptosis in this assay (Figure 1G, H). To test whether Cdk5 has a conserved role in mammals, we used mouse Min6 cells, which readily succumb to apoptosis when treated with tunicamycin (Supplementary Figure S2), a compound that inhibits protein glycosylation and cause stress in the ER 26. Knockdown of Cdk5 strongly suppressed tunicamycin-induced apoptosis, as assessed through TUNEL labeling (Supplementary Figure S2). Cdk5 levels did not change in response to Rh-1G69D expression (Figure 1U, V), suggesting that the protein is regulated by post-transcriptional mechanisms. In fact, Cdk5 activity is often regulated through its regulatory subunit, p35 (also known as Cdk5alpha)27. In a loss of function p35 (Cdk5alpha) background, the amount of apoptosis induced by Rh-1G69D expression was significantly reduced (Figure 1I, J), further confirming the role of Cdk5 in apoptosis.
To determine if the cdk5 knockdown condition suppresses apoptosis by reducing the overall stress levels in the ER, we labeled imaginal discs with the anti-ATF4 antibody. The degree of ATF4 induction in Rh-1G69D overexpressing eye discs was not affected by cdk5 knockdown (Figure 1K–M). We also assessed the extent of IRE1/XBP1 pathway activation, using the XBP1-EGFP reporter. Again, knockdown of cdk5 did not affect the degree of this ER stress reporter activation in response to Rh-1G69D expression (Figure 1N–P). These observations indicate that cdk5 mediates Rh-1G69D-induced apoptosis without affecting the overall levels of misfolded protein load in the ER. To further test if the ATF4 and IRE1/XBP1 pathways contribute to Rh-1G69D-induced apoptosis, we examined the degree of cell death in mutants that disrupt these pathways. In the loss of function ATF4 condition, crc −/− 32, the degree of Rh-1G69D-induced apoptosis was similar to those of the crc+ background (Figure 1Q, R). In the ire1 −/− mosaic clones, the degree of Rh-1G69D-induced apoptosis was increased (Figure 1S, T). Overall, these results show that ATF4 and IRE1 are not required for Rh-1G69D expression to induce apoptosis.
Independently, we performed a gene overexpression screen with Epgy2 lines 28 for modifiers of the gmr-Gal4 driven Rh-1 overexpression phenotype (Supplementary Figure S3). While a wild type Rh-1 transgene was used in this experimental setup, the system drives the expression of Rh-1 beyond the folding capacity of the imaginal disc cells, as indicated by the activation of ER stress reporters 5. We specifically screened 400 lines with insertions in the 3rd chromosomes that were associated with genes with annotated function and scored a total of six suppressors. Among these suppressors were expected ones, including a line associated with hrd1 (P{EPgy2}sip3[EY11980]), whose effect on the Rh-1G69D misexpression phenotype was independently validated in Supplementary Figure S1. Another expected suppressor line was P{EPgy2}th[EY00710], with P{EPgy2} element inserted upstream of the anti-apoptotic gene, Drosophila IAP1 (Diap1) 29, indicating that excessive apoptosis contributes to the Rh-1 overexpression phenotype.
We also identified an enhancer of the Rh-1 overexpression phenotype, EY02276, associated with the mekk1 locus. Previous studies have characterized mekk1 as an osmotic stress response gene that lies upstream of JNK and p38 kinases 30, 31. This line did not show any overexpression associated phenotype on its own, but enhanced the Rh-1 overexpression phenotype when co-expressed (Supplementary Figure S3). Conversely, the Rh-1G69D misexpression phenotype was suppressed in the mekk1ur-36 −/− background (Figure 2A–C). Upon inspection of imaginal discs, we found that the mekk1ur-36 −/− background almost completely suppressed apoptosis triggered by Rh-1G69D-overexpression (Figure 2D–F). To determine if mekk1 affects the overall levels of stress in the ER, we assessed the degree of XBP1-EGFP and ATF4 activation in eye imaginal discs overexpressing Rh-1G69D. We found no discernible difference between the mekk1+ and mekk1UR-36 −/− discs in the level of these ER stress reporters (Figure 2G–L), indicating that mekk1 specifically mediates the pro-apoptotic signaling response without affecting the degree of ER-stress. To further test the role of mekk1 in ER-stress-induced toxicity, we subjected mekk1 −/− adults to an independent assay, in which the flies were fed tunicamycin and their survival rate was monitored. While control wild type flies were vulnerable to this regimen, with only 15.7% of the flies surviving after 7 days of tunicamycin feeding, the survival rate of mekk1 −/− flies was much higher under identical conditions (57.8%). Three independent trials of this assay gave a statistically significant difference in the survival rate of mekk1 mutant flies (p=0.0062) (Figure 2M).
Figure 2
Figure 2
Drosophila Mekk1 is required for Rh-1G69D to trigger apoptosis
As previous studies have placed mekk1 genetically upstream of JNK 30, we examined the relationship between JNK, Mekk1 and Cdk5. For this, we exposed Drosophila S2 cells to thapsigargin (Tg), a SERCA inhibitor that is widely used to cause ER stress in cells32. Phospho-JNK appeared after 2 hours of Tg treatment (Figure 3A), and this induction of JNK phosphorylation was suppressed upon knockdown of cdk5 or mekk1 (Figure 3A), or when cells were treated with the Cdk5 inhibitor, roscovitine (Figure 3B). On the other hand, knockdown of other known mediators of the UPR, such as ire1, traf4, perk and atf6 had no discernible effects on JNK phosphorylation (Figure 3A). Among other stress conditions tested, H2O2 treatment generated a similar outcome (Figure 3C–F). H2O2's ability to induce JNK phosphorylation was significantly reduced in S2 cells pretreated with dsRNA targeting cdk5 or mekk1. While H2O2 treatment resulted in a more than six fold increase in phospho-JNK levels in control cells, cdk5 knocked down cells had on average only a two fold increase in phospho-JNK induction (Figure 3D)(n=3, p=0.018). Likewise, mekk1 knockdown reduced the extent of phospho-JNK induction in a statistically significant manner (Figure 3F)(n=3, p=0.0023).
Figure 3
Figure 3
Mekk1 and Cdk5 mediate JNK signaling activation in response to stress
Consistent with the results from S2 cells, Rh-1G69D misexpressing imaginal discs showed signs of JNK signaling activation, as assessed through the puc-lacZ reporter (Figure 3G–I). To test if JNK is required for ER-stress-induced apoptosis, we generated loss-of-function mosaic clones of the Drosophila JNK gene, basket. When Rh-1G69D was overexpressed in imaginal discs harboring basket −/− clones, the number of apoptotic cells as assessed through TUNEL labeling was significantly reduced, with the remaining apoptotic cells primarily within the basket+ mosaic clones (Figure 3J–L). We noticed that many apoptotic cells were found at the clonal boundaries. This property was also observed in mutant mosaic clones of dronc (Supplementary Figure S4), which is an essential initiator caspase for apoptosis33,34. These observations support the idea that ER-stress activates Cdk5/Mekk1-mediated JNK signaling to cause caspase-dependent apoptosis.
Using a phosphorylation site prediction program (http://scansite.mit.edu), we detected two consensus Cdk5 phosphorylation sites within the Drosophila melanogaster Mekk1 protein sequence, T157 and S1127. The putative phosphorylation sites within Mekk1were conserved in other Drosophila species, suggestive of its functional significance (Figure 4A). To test if Mekk1 is in fact phosphorylated by Cdk5, we generated antibodies directed against the putative phospho-residues (see Methods). Using one of these, an antibody directed towards the phosphorylated S1127 residue, we were able to detect Mekk1 phosphorylation by Cdk5 in vitro (Figure 4B). We also detected phosphorylation of this residue in cultured HEK293T cells transfected with flag-tagged Mekk1 (Figure 4C). Notably, the intensity of the phospho-S1127 band increased significantly in cells when Cdk5 was co-transfected, and further enhanced when those cells were stressed with H2O2 (Figure 4C lanes 3, 4). On average, the degree of Mekk1 phosphorylation increased more than three fold after H2O2 treatment (Figure 4C, n=3, p=0.0003). We confirmed that this band corresponds to phospho-S1127, as the signal did not appear when the Mekk1 S1127 residue was mutated (Figure 4D lane 5, 6). Furthermore, the anti-phospho-Mekk1 failed to detect any band when the immunoprecipitate was treated with the lambda phosphatase (Figure 4E). Moreover, the two proteins physically interacted, as evidenced by co-immunoprecipitation assays. Interestingly, the interaction was enhanced when the cells were pre-treated with H2O2 (Figure 4F). Taken together, these genetic and biochemical experiments support the idea that Cdk5 and Mekk1 form a pathway to activate JNK signaling in response to ER-stress.
Figure 4
Figure 4
Cdk5 phosphorylates Mekk1
To test if this pro-apoptotic signaling pathway is also relevant to an age-dependent disease process, we turned to the Drosophila model for ADRP, where an endogenous mutant allele of the Rh-1 gene, ninaEG69D, causes late-onset retinal degeneration phenotype associated with ER stress9, 10. To track the course of retinal degeneration in live flies, we used the ninaEG69D/+ condition combined with a Rh-1>GFP reporter 35. Nearly 90% of ninaEG69D/+ flies lost the regular ommatidial array by day 28 after eclosion, indicative of age-related retinal degeneration. Those ninaEG69D/+ flies in a mekk1ur-36 −/− background showed a delayed course of retinal degeneration, with only about half of the examined flies with disrupted Rh-1>GFP patterns (Figure 5A). Knockdown of cdk5 in the photoreceptors also delayed the course of retinal degeneration to a similar degree (Figure 5F). This result was further validated through tangential sections of 20 day old fly retina. Wild type flies showed regular ommatidial arrays (Figure 5B), while the ninaEG69D/+ retina by this age showed disorganized ommatidia (Figure 5C, G). This phenotype was largely rescued in the backgrounds of mekk1ur-36 −/− (Figure 5D), or in cdk5 knockdown conditions (Figure 5H).
Figure 5
Figure 5
The course of late onset retinal degeneration of ninaEG69D/+ flies is delayed upon knockdown of Cdk5, or in the mekk1ur-36−/− background
These results indicate that the pro-apoptotic ER-stress response mediated by mekk1 and cdk5 are relevant to understanding age-related photoreceptor degeneration in ADRP. Moreover, our results suggest that Cdk5/Mekk1/JNK forms a pathway that is independent of those UPR branches. While it is unclear what lies upstream of Cdk5 in our experimental system, we note that among the previously characterized Cdk5 activating signals include ROS, calpains and Cam kinase II23, 25, 36, 37, which have been also associated with ER-stress 16, 38. Thus it is possible to envision a model where chronic proteotoxicity in the ER sends Cdk5 activating signals to the cytoplasm, perhaps via ROS or Ca2+ mediated signaling. Once Cdk5 is activated, it may send pro-apoptotic signals to the nucleus through the Mekk1/JNK pathway (Supplementary Figure S5).
Many terminally differentiated cells without regenerative potential are known to acquire resistance to apoptosis during differentiation. In Drosophila, such apoptotic resistance can be attributed to the epigenetic silencing of major pro-apoptotic gene loci during development 39. A recent study showed that one of the consequences of stress-induced Mekk1-signaling is to induce the expression of genes that are normally silenced through epigenetic mechanisms31. Based on these observations, we think it is possible that terminally differentiated photoreceptors may have their pro-apoptotic loci in heterochromatin-like states, and stress-induced Cdk5/Mekk1 pathway contributes to neurodegeneration by restoring those loci to an open chromatin state, an idea that needs to be tested through future studies.
Supplementary Material
Acknowledgements
We thank Ed Giniger, Kunihiro Matsumoto, Masayuki Miura, the VDRC and Bloomington stock centers for reagents, Edith Robbins, Michele Pagano and Ester Zito for technical advice, and David Ron for discussions and critical comments on the manuscript. This work was supported by the National Institutes of Health grant R01EY020866. H.D.R. is an Ellison Medical Foundation New Scholar.
Footnotes
Author Contributions M. K and H.D.R. designed the experiments. J.C. carried out the EP screen. All other experiments were performed by M. K. H.D.R. wrote the paper and all authors read and edited the manuscript.
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