On the role of ras and BRF1 in the regulation of ARE-dependent mRNA-turnover

Stability of the mRNAs of cytokines, chemokines, growth-factors, protooncogenes
and others are regulated via an AU-rich element (ARE) in their 3’
untranslated region (UTR). Upon activation of signal transduction pathways,
these mRNAs, which are short-lived under resting conditions, become stabilised.
AU-rich element binding proteins (AUBPs) are directly involved in this process
as they either promote decay (TTP, BRF1, AUF1, KSRP) or stabilisation (HuR) of
the mRNA by binding to the ARE.
In this work, the infl uence of the small GTPase ras and three of its downstream
signalling pathways on the stability of the IL-3 ARE-mRNA was investigated.
Work in our laboratory has identifi ed BRF1 as an AUBP responsible for IL-3 AREmRNA
decay. Therefore the effects of these pathways on BRF1 activity was also
studied.
Ras, as the most upstream signalling protein, not only stabilises the IL-3 AREreporter
transcript but was also able to completely inhibit the activity of BRF1,
indicating that ras activates one or several pathways that are able to inactivate
BRF1. However, PI3-K and raf, two downstream targets of ras were not able to
overcome the induction of ARE-mRNA decay by BRF1, although they were able
to stabilise the IL-3 ARE-reporter on their own. Even in combination, PI3-K and
raf were not able to inhibit BRF1.
Two downstream targets of PI3-K, PKB and rac, were able to stabilise the reporter
but did not antagonise the decay-inducing activity of BRF1. An active p38
pathway was necessary for rac to stabilise the reporter as shown by experiments
using a dominant negative form of p38. However the p38 pathway alone was not
suffi cient, as an activated form of MEK6 did not stabilise the IL-3 ARE-reporter,
indicating that rac needs to activate at least two downstream pathways in order
to stabilise ARE-mRNA.
BRF1 has been found to harbour consensus sequences for phosphorylation by
PKB and MK2, a downstream target of p38. In a co-transfection experiment it was
possible to demonstrate that MK2 and PKB together were able to inhibit BRF1
activity.
Mass-spectrometry on recombinant BRF1 in vitro phosphorylated by PKB
revealed serine 92 as the PKB phosphorylation site. Therefore a mutant was
constructed in which serine 92 was replaced with alanine, thereby preventing
phosphorylation at this site, and tested alongside wild-type BRF1 in an in vitro
decay assay. As expected the wild-type BRF1 protein induced a very fast decay,
but surprisingly PKB phosphorylated BRF1 was completely inactivated, which is
in contradiction to the cellular decay system. In addition the mutant BRF1S92A,
which was as active as the wild-type BRF1 in promoting decay, was not inactivated
by PKB phosphorylation, proving the importance of serine 92 in regulation of
BRF1 activity by PKB.
For future work it would be of interest to obtain additional in vivo data regarding BRF1 regulation by PKB and to investigate the role of MK2, as it appears to
participate in in vivo decay but not in the in vitro decay system.