Rheb-T23M and E40K drive mild endoplasmic reticulum stress
through increased protein synthesis
In a recent study (Jianling Xie et al., 2020), we showed that certain
mutations in Rheb (which arise in human cancers) are able to drive
hyperactive mTORC1 signalling in mouse or human cells.
As mTORC1 is well known to promote multiple steps in mRNA translation
(protein synthesis), we assessed whether these Rheb mutants can drive
increased protein synthesis. To do this we employed surface sensing of
translation western blot assay (SUnSET-WB (Schmidt, Clavarino, Ceppi, &
Pierre, 2009)). In the SUnSET-WB technique cells are treated with a low
concentration of puromycin (1 µM), a compound which acts as a structural
analogue of aminoacyl-tRNA (specifically tyrosyl-tRNA) and can therefore
be incorporated into nascent polypeptides. Incorporated puromycin can
then be detected by western blot with puromycin-specific antibodies.
Thus SUnSET-WB is a radioactive-free assay to measure rates of protein
synthesis. For our initial experiments, we elected to utilise HEK293
cells as they are both a common cell line used for cell signalling
studies, and ones in which we have previously shown Rheb mutations drive
constitutive mTORC1 signalling. HEK293 cells transiently transfected
with vectors encoding FLAG-Rheb[WT], [T23M], [Y35N] or
[E40K] or an empty vector (EV) were transferred to Dulbecco’s
phosphate buffered saline (D-PBS) for 30 min prior to the addition of 1
µM puromycin for an additional 30 min. One well of untransfected cells
was pre-treated with 50 µg/mL of cycloheximide, a potent inhibitor of
protein synthesis, for 30 minutes prior to the addition of puromycin to
provide a ‘negative control’ for any immunostaining that is not due to
ongoing protein synthesis. Cell lysates were then harvested for western
blot analysis. Rheb[T23M] and [E40K] each stimulated a large
increase in puromycin incorporation compared to either Rheb[WT] or
EV (Fig. 1a; quantified in Fig. 1b). Interestingly, despite
Rheb[Y35N] being known to drive hyperactive mTORC1 signalling, it
did not increase puromycin incorporation, in line with the fact that
Rheb mutants differ in their downstream effects (Jianling Xie et al.,
2020).
It has been shown that increases in overall protein synthesis can
overload the protein folding capacity of the endoplasmic reticulum (ER)
resulting in ER stress and activation of the unfolded protein response
(Sriburi et al., 2004). This process has been observed in response to
increased mTORC1 activity (Appenzeller-Herzog & Hall, 2012; Dong et
al., 2015) and results in an expansion of the ER and therefore increased
protein folding capacity (Shaffer et al., 2004; Sriburi et al., 2004; M.
Wang & Kaufman, 2016). We hypothesised that Rheb-mutants may drive mild
ER stress resulting in increased ER volume and protein folding capacity.
To test this, we first studied several proteins involved in both the
ATF4 arm of the UPR and proteins involved in protein folding. Cells
stably expressing plasmids encoding Rheb[WT] or mutants of Rheb
showed increased expression of ATF4 compared to the empty vector (Fig.
1c). Interestingly, there was no significantly greater change in ATF4
protein expression in cells expressing Rheb mutants compared to WT.
However, Rheb[T23M] and [E40K] did promote an increase, or
tended to cause an increase, in classical UPR markers or ER resident
proteins including PERK, BiP/Grp78, IRE-1α, PDI and ERO1-1α (Fig. 1c;
quantified in Supplementary Figure 1). Calnexin did not change. There
was also an increase in the protein folding markers ERO1-Lα (Fig. 1c;
quantified in Supplementary Fig. S1).
To assess whether these changes reflected increased levels of the
corresponding mRNAs, we performed RT-qPCR for the mRNAs encoding BiP
(HSPA5 ; Fig. 1d), PDI (PDI ; Fig. 1e), IRE1α (ERN1 ;
Fig. 1f), and ATF4 (ATF4 ; Fig. 1g) whose levels were increased by
mutant Rheb expression. Increases in mRNA encoding both UPR and protein
folding proteins correlated with protein increases with the notable
exception of ATF4 mRNA which was significantly higher in cells
expressing Rheb mutants compared to both the EV and Rheb[WT] (Fig.
1d-g). To determine if these observed changes are associated with an
increase in ER volume, we performed immunofluorescence on HEK293 cells
stably expressing Rheb mutants or WT as well as an empty vector. To
image the ER, we chose to probe with an anti-calnexin antibody as
calnexin is an ER surface protein and there was no significant change in
the level of calnexin protein with the different Rheb mutants (Fig. 1b)
(so that any alterations seen in the extent of the ER would be
independent of changes in its overall levels). Volume was calculated
based on a β-actin counter stain. Both Rheb[T23M] and [E40K]
promote a significant increase in ER volume compared to Rheb[WT] and
EV. Rheb[Y35N] did not promote an increase in ER volume (Fig. 2a;
quantified Fig.2b). These data suggest the Rheb[T23M] and [E40K]
mutants can each promote increase protein synthesis which, in turn,
drives a mild ER stress resulting in increased ER volume and concomitant
protein folding capacity.
These data for HEK293 cells prompted us to extend our studies to CHO
cells, the dominant type of cells used for in industry for producing
recombinant proteins, particularly as an expanded ER is reported to
enhance the ability of CHO cells to produce secretory recombinant
proteins (Budge et al., 2020).