Among other functions, macroautophagy (hereafter autophagy) is a critical regulator of cell survival and death. For example, we have previously shown that basal autophagy affects cell fate in response to pro-death stimuli.^1,2 Moreover, some cancer cells are dependent on basal levels of autophagy for survival.³ While many mechanisms that control the induction of autophagy have been characterized, the regulation of basal autophagy is incompletely understood. It may depend on factors that direct transcription, such as TFEB⁴ and the FOXO family of transcription factors, which control the basal expression of autophagosomal and lysosomal genes. To find genes that regulate basal autophagy, we conducted a synthetic lethality/viability screen for kinases that affect growth/viability in cells when under stresses that promotes reliance on autophagy, as detailed in our recent publication in EMBO Reports.⁵ We identified the serine-threonine kinase RIP1 (RIPK1) as a gene whose knockdown is highly selected against during growth in normal media where autophagy is not critical, but selected for under conditions where increased autophagy is advantageous.⁵

In addition to our kinase screen, we found RIP1 shRNA expression was selected against under normal growth conditions in 7 other cell lines in previously shRNA screens conducted in our lab. Upon perusing the literature, we found this to be consistent with an analysis of multiple genome-wide shRNA lethality screens conducted by the HTS Core Facility at Memorial Sloan- Kettering Cancer Center (MSKCC), which indicated that RIP1 shRNAs were selected against in 9 of 12 cancer cell lines where such screens were conducted.⁶ This consistently observed negative selection of RIP1 shRNAs under normal growth conditions underscored to us the significance that the presence of RIP1 shRNAs were selected for when autophagy was artificially limited in 67NR cells (these cells require autophagy for growth) or when these cells were starved in Earle's Balanced Salt Solution (EBSS). Upon further investigation, we found that the acute knockdown of RIP1 led to a large increase in basal autophagic flux in multiple cell types. Efficient RIP1 knockdown also led to other phenotypes that appeared to be cell-type specific, such as reduction of cell growth, the formation of large multinucleated cells, and increased spontaneous cell death.

We found that RIP1 knockdown led to increased expression of ATG (autophagy-related) and lysosomal genes, consistent with effects on transcriptional regulation of autophagy. RIP1 knockdown cells had reduced phosphorylation of TFEB at serine 142, which phosphorylation status has been shown to negatively regulate its transcriptional enhancement of autophagy and lysosomal genes.⁴ RIP1 knockdown led to increased nuclear localization of TFEB and furthermore activated a promoter containing 4 tandem TFEB recognition elements (4xCLEAR), indicating that RIP1 was likely suppressing TFEB-mediated transcription. When we examined indicators of the activities of the 2 kinases that are known to phosphorylate serine 142 of TFEB (MAP kinase ERK2 and mTORC1), basal ERK phosphorylation was significantly reduced in RIP1 knockdown cells whereas the phosphorylation of p70 S6 kinase, an mTORC1 substrate, did not differ upon RIP1 knockdown. Overexpression of RIP1 was sufficient to activate ERK and importantly, expression of a constitutively active MEK (the direct upstream activator of ERK) or a TFEB shRNA was sufficient to prevent RIP1 knockdown from inducing autophagy. Thus we conclude that RIP1 represses basal autophagy due to its ability to negatively regulate the TFEB transcription factor through its activation of ERK. Partial knockdown of RIP1 increased the sensitivity of MCF10A cells to FasL but protected from TRAIL-induced cell death in a manner dependent on increases in autophagy, consistent with our previous observations that higher basal autophagy selectively potentiates Fas ligand mediated cell death while inhibiting TRAIL-mediated cell death.¹

RIP1 has previously been shown to play multiple roles related to cell death signaling. In addition to its ability to activate MAP kinases, such as JNK, p38, and ERK, RIP1 is also important for the activation of pro-survival signals through NF-κB-mediated transcription. RIP1 also has multiple pro-death functions. It is essential for induction of regulated necrosis triggered by death receptor agonists (also referred to as necroptosis) through its interactions with RIP3, which leads to the activation of MLKL.⁷ RIP1 has also been reported to contribute to caspase-8 activation downstream of death receptors, and so it contributes not only to necrosis, but also apoptosis. To the list of processes and signaling pathways by which RIP1 affects cell death, our study now adds another: the control of cellular sensitivity to pro-death stimuli by regulating basal autophagy via modulation of ERK activity (see Fig. 1).

Due to its effects on multiple cell death and survival pathways, we still do not yet understand exactly why acute loss of RIP1 leads to growth suppression or cytotoxicity. Further experiments utilizing RIP1 mutants that interact with specific pathways may be fruitful in identifying which pathways are important for these effects. Although we do not currently have any data to support this conjecture, it is possible that in some cell lines the resulting high levels of autophagy over prolonged periods of time are directly toxic to the cells. Or it may be possible that RIP1 binding partners (e.g. RIP3) are hyperactivated in the absence of RIP1, and result in toxicity.

Basal autophagy is negatively regulated by RIP1

All authors

Michael J Morgan

https://doi.org/10.1080/15384101.2015.1080978

Published online:

23 October 2015

Figure 1. Downstream of TNF Receptor −1 and other death receptors, RIP1 is important for the activation of NF-κB and MAP kinase pathways, and is involved in the formation and regulation of secondary complexes that are responsible for resultant apoptosis and regulated necrosis. Our recent study suggests that RIP1 also represses basal autophagy via modulation of ERK activity and regulation of TFEB.

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