http://orcid.org/0000-0002-1167-2676
ABSTRACT
In neurons, autophagy counteracts consequences of aging. It is therefore of interest how basal rates of macroautophagy/autophagy can be controlled independently of metabolic stress. We recently investigated the regulation of basal, starvation-independent autophagy by Acn/Acinus, a multifunctional nuclear protein with proposed roles in apoptosis, alternative RNA splicing, and basal autophagy. We found that Acn is stabilized by phosphorylation of the conserved serine 437. The phosphomimetic AcnS437D mutation causes no overt developmental phenotypes, but significantly elevates levels of basal autophagy and extends life spans. An RNAi screen identified Cdk5 as a kinase targeting S437, a role confirmed by gain- and loss-of-function mutants of Cdk5 or its obligatory cofactor Cdk5r1/p35. Flies lacking Cdk5 function display reduced basal autophagy and a shortened life span. Both of these phenotypes are suppressed by the phosphomimetic AcnS437D mutation, indicating that phosphorylating serine 437 of Acn, and thereby maintaining basal levels of autophagy, is critical for Cdk5's function in maintaining neuronal health.
Improvements in proteomics approaches have provided an astonishing treasure trove of data detailing protein levels and their post-translational modifications (PTM) in different cell types and species. Understanding the physiological relevance of such modifications, however, requires functional experiments. Recently, we examined the role of one such PTM that has been observed in samples from mammalian and Drosophila cells: the phosphorylation of the conserved serine 437 of Acn.
Acn is a nuclear protein that has been implicated in diverse roles in apoptosis, alternative splicing and autophagy. In previous work, we showed that levels of Drosophila Acn are regulated at the protein level, in part by Akt1-dependent phosphorylation which inhibits degradation of Acn by the CASP3 homolog Dcp-1 (). CASP3 cleaves ACIN1/Acinus in mammalian cells undergoing apoptosis, but in Drosophila we detect this cleavage in non-apoptotic neurons. Importantly, preventing this cleavage elevates Acn levels and increases basal, starvation-independent autophagy. Thus, we were curious whether phosphorylation at serine 437 also alters Acn level and function.
A phospho-specific antibody detects widespread, dynamic phosphorylation of serine 437 in cells of the developing eye and other tissues. To test the physiological relevance of this PTM, we replaced the endogenous Acn protein with nonphosphorylatable AcnS437A or phosphomimetic AcnS437D, either one expressed under control of the endogenous Acn promoter. Despite unchanged RNA levels, protein levels are significantly increased by the phosphomimetic S437D mutation and decreased by the alanine substitution. This suggested that serine 437 phosphorylation stabilizes Acn. Nevertheless, both of these mutant Drosophila lines lack overt developmental phenotypes.
Differences became obvious, however, when we compared their levels of autophagy. In developing eyes and fat bodies, AcnS437D supports elevated numbers of Atg8-positive autophagosomes, increased autophagic flux and enlarged lysosomes, when compared to AcnS437A or wild-type Acn. Similar to other fly lines with elevated autophagy levels, AcnS437D flies exhibit improved starvation resistance and, when fed well, their median life span is extended approximately 50% compared to the wild type.
Motivated by this substantial impact of Acn serine 437 phosphorylation, we set up an RNAi screen to identify the responsible kinase(s). As serine 437 is flanked by 2 prolines, we focused on the subset of proline-directed protein kinases. The screen made use of the externally visible “rough-eye” phenotype caused by eye-specific overexpression of wild-type Acn. We reasoned that this “rough-eye” phenotype should be ameliorated once Acn level and function are reduced by eliminating the stabilizing effect of serine 437 phosphorylation. This prediction is matched by the knockdown of 2 kinases, Cdk5 and p38b MAP kinase (MAPK).
Using gain- and loss-of-function alleles of Cdk5, or its obligatory co-activator Cdk5r1/p35, we found that the Cdk5-Cdk5r1/p35 complex is responsible for most of Acn S437 phosphorylation in imaginal discs. Furthermore, AcnS437A overexpression phenotypes, unlike those of wild-type Acn, are not enhanced by Cdk5r1/p35 co-expression. This indicates that serine 437 is not just one of many sites on Acn targeted by Cdk5, and instead is critically required for their genetic interactions. In a null allele of p38b MAPK, by contrast, phosphorylation of Acn S437 is not diminished, and the interaction between p38b MAPK and Acn overexpression is independent of phosphorylation at serine 437. Thus, whereas p38b MAPK may target different Acn residues or interact indirectly with Acn, the Cdk5-Cdk5r1/p35 complex directly phosphorylates serine 437 as we also confirmed by in vitro phosphorylation assays.
Cdk5 is a member of the family of cyclin-dependent kinases, but unlike other members, is not required for the regulation of mitosis. Instead Cdk5 is prominently expressed in post-mitotic neurons where its activation has been observed in the context of multiple neurodegenerative diseases. The findings that phosphorylation of Acn regulates basal, starvation-independent autophagy suggests that Cdk5-Cdk5r1/p35 activity is necessary to maintain basal autophagy. This was confirmed in Cdk5r1/p35 mutant eye discs, which display reduced numbers of autophagosomes and elevated levels of the autophagy substrate ref(2)P/p62. Importantly, the phosphomimetic AcnS437D mutant restores wild-type autophagy levels in Cdk5r1/p35 mutants, indicating that Acn is a key substrate of Cdk5-Cdk5r1/p35 for promoting autophagy. This conclusion was further substantiated by our finding that phosphomimetic AcnS437D restores the shortened life span of Cdk5r1/p35 mutants to near wild-type levels.
Given the link of Cdk5 activation to neurodegenerative diseases, we tested corresponding Drosophila disease models for elevated Acn serine 437 phosphorylation. Interestingly, Cdk5-Cdk5r1/p35-dependent serine 437 phosphorylation is induced by polyQ-htt (Huntingtin) expression. Similarly, Acn phosphorylation is elevated by other polyQ proteins linked to ataxias and also by Aβ42, a hallmark of Alzheimer disease, but not SNCA/alpha synuclein or SOD1, 2 proteins with links to Parkinson disease and amyotrophic lateral sclerosis, respectively.
These observations prompt many follow-up questions, to name just 2: What is the mechanism by which Acn promotes autophagy? Phosphorylation of the corresponding serine in mammalian ACIN1 has been reported, but are the functional consequences conserved as well?
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.