We have used whole-genome microarrays to identify genes regulated during the starvation response in Drosophila, and also compared gene expression changes between control, Atg7
mutant larvae. Based on a gene ontology analysis, genes involved in catabolic processes such as proteasomal degradation and amino acid catabolism were more significantly upregulated in starved autophagy mutants, which is likely a compensatory reaction. On the other hand, genes required for DNA replication were specifically downregulated. Mitotic tissues continue to grow and divide even in Drosophila larvae deprived of all nutrients.36
Repression of genes required for DNA replication in autophagy mutants suggests that energy-consuming DNA replication processes are strongly inhibited in completely starved autophagy mutants, presumably because polyploid cells are not able to supply nutrients to support diploid cell divisions in the absence of autophagy.
We detected increased transcription of most Atg
genes upon starvation, and an even higher upregulation was seen for most genes in fat bodies dissected from starved vs. fed animals. The most highly induced gene was Atg8a
in all cases, in agreement with previous studies that showed the importance of Atg8
induction during autophagy in various models.1,12,15
It is important to note that Atg8b
is mostly expressed in adult testis, and its very low level larval expression is restricted to the fat body.37
In line with this, mutation of Atg8a
completely blocks starvation-induced autophagy in the fat body confirming that Atg8b
expression is unable to sustain autophagy in this setting, although Atg8b
was also upregulated 118-fold during starvation in the fat body.21
Induction of Atg8a
is likely necessary to make up for the degradation of half of its protein products involved in each autophagosomal cycle. Members of the Atg9 cycling complex showed the strongest average upregulation of the functional groups of Atg proteins. We speculate that the sudden induction of autophagy in polyploid larval Drosophila tissues requires a lot of membrane to sustain the high level of autophagosome generation, explaining the increased transcription of genes whose products are required for membrane transport.
In this work we chose Rack1 (receptor of activated protein kinase C 1) for further analysis. Rack1 was induced in whole animals and in dissected fat bodies during starvation, and loss of Rack1 impaired both starvation-induced and basal autophagy, while it had no effect on developmentally programmed autophagy of the fat body. These findings indicate that Rack1 is involved in multiple but not all types of autophagy.
Autophagosomes generally have a short half-life of 5–10 min, which is the most likely explanation why basal levels of autophagy are very difficult to visualize in most tissues.38
In contrast, autophagosomes appear in high numbers during starvation. The autophagosomal compartment was reduced in Rack1
mutants which was rescued completely by transgenic expression of Rack1
, and Rack1 was at least transiently associated with phagophore assembly sites and autophagosomes, altogether suggesting that Rack1 is required for efficient autophagosome formation. As we observed no defects in cell size/cell growth, DNA polyploidization or lipid droplet accumulation in Rack1
loss of function cells, the autophagy defect seems to be a highly specific phenotype and not just a consequence of general problems with normal cellular functions.
Rack1 is an evolutionarily conserved guanine nucleotide-binding scaffold protein with a WD40-repeat: 77% (243/315) of the amino acid residues are identical and 87% (275/315) are similar between Drosophila Rack1 and human GNB2L1. A proteomic study of Atg complexes found that GNB2L1 interacts with human homologs of Atg1, Atg4, Atg14 and Atg18.39
Although GNB2L1 was not classified as a high-confidence interacting partner, these data support our hypothesis that Rack1 may act as a scaffold, transiently binding multiple Atg proteins at phagophore assembly sites to promote maximal activity. In line with that, it is interesting to note that the potential interacting partners of human Rack1 include a member from all four Atg protein complexes. Further biochemical studies such as co-immunoprecipitation experiments are necessary to verify this hypothetical mechanism.
In addition, half of the Rack1-positive dots localized to glycogen particles, and loss of Rack1 prevented the proper formation of glycogen stores in larval fat body cells. The high degree of colocalization with GSK-3B strongly suggests that Rack1 is associated with a pool of GSK-3B that promotes glycogen synthesis. The known interacting partners of Rack1 include three subunits of AMPK (AMP-activated protein kinase): PRKAA1, PRKAA2 and PRKAB2. The β-subunit of AMPK has a glycogen-binding domain that targets a pool of this kinase to bind the surface of the glycogen particle. Activated AMPK turns on catabolic processes to generate ATP, and it also inhibits glycogen synthesis through direct phosphorylation of glycogen synthase.40
We hypothesize that a signaling complex containing Rack1, GSK-3B and AMPK assembles on glycogen particles to regulate the rate of synthesis through phosphorylating different amino acid residues of glycogen synthase, with AMPK being responsible for targeting this complex to glycogen. In this scenario, the activity of multiple kinases including AMPK and GSK-3B would determine the rate of glycogen synthesis. These interactions may be less relevant for Rack1’s role in autophagy for a number of reasons. First, GSK-3B never colocalized with Atg8a, and overexpression of GSK-3B actually inhibits starvation-induced autophagy (our unpublished observation). Second, the exact role of AMPK in starvation-induced autophagy of the fat body is not clear: mutation of SNF4Aγ
encoding a regulatory subunit of AMPK has been shown to lead to impaired autophagic responses in mutants, but a more recent report suggested that AMPK mutants show a persistent starvation phenotype that may interfere with further induction of autophagy by additional experimental starvation.41,42
In line with that, none of the more than 10 different transgenic RNAi and dominant-negative AMPK lines we tested showed any suppression of starvation-induced autophagy in clonal analysis (our unpublished observations), potentially supporting the hypothesis that the autophagy phenotype of AMPK mutants may not be cell autonomous in the Drosophila fat body.
Rack1 was originally described as a cytoplasmic receptor for activated protein kinase C (PKC). The structure of Rack1 resembles that of the β-subunit of heterotrimeric G proteins. The individual WD40 repeats can simultaneously bind to different proteins, making Rack1 a candidate platform for integrating several signaling pathways. Rack1 was shown to physically interact with β-integrin, various kinases including PKC, AMPK, GSK-3B and Src, protein phosphatase 2A, focal adhesion components, and even ribosomes. Indeed, Rack1 plays a role in a wide range of processes including cell adhesion and migration, cell survival and translation.33,43
Recent findings showed that Rack1 promotes and is required for progression of several types of cancers, and its increased expression predicts poor clinical outcome for breast cancer patients.44-48
Here we have shown that Drosophila Rack1 is also involved in the autophagic response to starvation, potentially acting again as a scaffold protein during the formation of autophagosomes. In addition, Rack1 is necessary for the proper generation of glycogen particles in the larval fat body, likely through recruiting Shaggy/GSK-3B to promote glycogen synthesis. Taken together, we have demonstrated that novel roles in autophagy and glycogen synthesis must be added to the already diverse list of functions for Rack1.