Rheb mutants drive constitutive mTORC1 signalling in CHO
cells
Enabling cells to produce more protein is highly beneficial for the
efficient production of therapeutic proteins such as monoclonal
antibodies (mAbs). mAbs are most commonly produced from Chinese Hamster
Ovary (CHO) cells (Kunert & Reinhart, 2016; Sharker & Rahman, 2020).
Given that Rheb mutants increase protein synthesis and enhance ER
capacity in HEK293 cells, we hypothesised that these mutants, when
expressed in CHO cells, may also result in increased secretory mAb
titres. For our initial experiments we utilised our previously reported
CHO cell lines that stably expresses either the secreted luciferase
reporter Gaussia Luciferase (GLuc) or Firefly Luciferase (FLuc).
As it was first important to confirm that Rheb mutants can also drive
hyperactive mTORC1 signalling in CHO cells, we co-transfected them with
vectors encoding Rheb mutants and with vectors encoding FLAG-TSC1/2.
Here we used both GLuc-CHO and FLuc-CHO cells grown in monolayer in
fully supplemented Ham’s F12 medium. Cells were then starved of serum
for 16 h and then transferred to D-PBS for 1 h prior to lysates being
harvested for immunoblot analysis. mTORC1 activity was assessed by
monitoring the phosphorylation of the well-characterized mTORC1
substrate, S6K1, at Thr389, rpS6 at Ser240 and Ser244 (a substrate of
S6K1) and of 4E-BP1 at Thr37, Thr46 and Ser65 (all direct substrates of
mTORC1). Phosphorylation of all effectors were slightly elevated in
cells expressing Rheb[WT] compared to cells that received EV and, as
expected, was almost completely abolished in cells that were
co-transfected with FLAG-TSC1/2 in both GLuc-CHO (Fig. 3a) and FLuc-CHO
cells (Fig. 3b).
In contrast, cells expressing Rheb[T23M] or [E40K] showed higher
levels of phosphorylation of S6K1, rpS6 and 4E-BP1, even in the presence
of FLAG-TSC1/2, suggesting these mutants drive hyperactivation of mTORC1
(i.e., which is insensitive to inhibition by TSC) in GLuc-CHO (Fig. 3a)
and FLuc-CHO (Fig. 3b) cells. These data are consistent with our
findings for expression of these mutants of Rheb in human cells
(Jianling Xie et al., 2020).
We next sought to determine the effect of Rheb mutations on cell
proliferation. To do this, GLuc-CHO cells were allowed to proliferate
for 7 days while being counted on a haemocytometer every day in order to
generate a cell growth curve. None of the Rheb mutants had any effect on
cell proliferation when GLuc-CHO cells were grown in fully supplemented
medium or medium supplemented with 0.5% FBS (Fig. 3c, Supplementary
Fig. S2a). However, when GLuc-CHO cells were grown in medium
supplemented with 1% FBS, Rheb[T23M] promoted both an increased
rate of proliferation as well as supporting an increased total cell
number (Supplementary Fig. S2b). This was even more pronounced when
cells were grown in the absence of FBS with Rheb[T23M] promoting a
marked increase in the rate of proliferation (Fig. 3d). To confirm these
results, BrdU incorporation assays were performed in order to more
directly quantify the effect of Rheb[T23M] on cell proliferation.
Consistent with our findings for HEK293 cells (Jianling Xie et al.,
2020), Rheb[T23M], [Y35N] and [E40K] each drove increased
cell proliferation in both GLuc-CHO and FLuc-CHO cells compared to both
Rheb[WT] and EV (Fig. 3e/f).