Biochem. J. (2008) 413, e7–e9 (Printed in Great Britain)
e7
doi:10.1042/BJ20081244
COMMENTARY
Phosphorylation of Argonaute proteins: regulating gene regulators
Sabine RÜDEL and Gunter MEISTER1
Center for integrated protein Sciences Munich (CIPSM), Laboratory of RNA Biology, Max–Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
Members of the Ago (Argonaute) protein family are the mediators of small RNA-guided gene-silencing pathways including
RNAi (RNA interference), translational regulation by miRNAs
(microRNAs) and transcriptional silencing. Recent findings by
Zeng et al. in this issue of the Biochemical Journal demonstrate
that Ago proteins are post-translationally modified by phosphorylation of Ser387 . Mutating Ser387 to alanine leads to reduced localization of human Ago2 to cytoplasmic P-bodies (processing
bodies), cellular sites where RNA turnover and, at least in part,
miRNA-guided gene regulation occurs. Zeng et al. further show
that a member of the MAPK (mitogen-activated protein kinase)
signalling pathway phosphorylates Ago2 at Ser387 , suggesting
that Ago2-mediated gene silencing might be linked to distinct
signalling pathways.
Small regulatory RNAs such as miRNAs (microRNAs), siRNAs
(short interfering RNAs) or piRNAs (Piwi-interacting RNAs) are
expressed in all higher eukaryotes and regulate gene expression
at the level of transcription, translation or mRNA stability
[1–3]. siRNAs as well as miRNAs are processed from ds
(double-stranded) precursor RNAs to mature small RNAs of
approx. 18–25 nt in size. This process involves the RNase III
enzyme Dicer and generates short dsRNA intermediates with characteristic 5 phosphate groups and 2 nt 3 overhangs (Figure 1).
Such intermediates are subsequently unwound and one strand is
incorporated into RNA–protein complexes giving rise to the guide
strands of siRNAs or mature miRNAs. siRNA-containing protein
complexes are termed RISCs (RNA-induced silencing complexes)
whereas miRNA–protein complexes are termed miRNPs. The
biogenesis of piRNAs is currently only poorly understood and
presumably involves single-stranded RNA precursors. piRNPs
(piRNA-containing protein complexes) have not as yet been
isolated and functionally analysed [2].
Ago (Argonaute) proteins are at the heart of small RNAcontaining silencing complexes and are therefore considered as
key proteins in all gene-silencing pathways known thus far [4,5].
Ago proteins are characterized by PAZ (Piwi, Argonaute and
Zwille) and PIWI domains. Structural analyses have implicated
the PAZ domain in binding the 3 end of small RNAs. Moreover,
another domain, termed the MID domain, has been identified
recently [6,7] and it has been demonstrated that the MID domain
specifically anchors the 5 end of small RNAs. The structure of
the PIWI domain is highly homologous with that of RNase H and
indeed it has been shown that some PIWI domains cleave target
RNAs that are complementary to Ago-bound small RNAs [6–8].
In humans, eight Ago proteins exist, which can be subdivided
into the Ago (Ago1, Ago2, Ago3, Ago4) and the Piwi subfamily
(Hiwi1, Hiwi2, Hiwi3, Hili) [4,5]. The expression of Piwi
subfamily members is restricted to the germline, where they
associate with piRNAs and most probably function in silencing
mobile genetic elements. The Ago subfamily is ubiquitously
expressed and the individual members are involved in miRNAor siRNA-guided gene-silencing pathways. Interestingly, only
human Ago2 contains endonucleolytic activity although the
postulated catalytic residues Asp587 , Asp669 and His807 are conserved in other human Ago proteins as well. It is therefore
tempting to speculate that other cellular mechanisms such as posttranslational modification may contribute to Ago function.
The study by Zeng et al. [9] in this issue of the Biochemical
Journal has analysed Ago protein phosphorylation and its
implications for Ago function [9]. Using in vivo labelling as well
as MS approaches, the authors find that tagged human Ago2 is
phosphorylated at Ser387 . Further analysis revealed that the phosphorylation signal is no longer detectable when Ser387 is mutated to an alanine residue (S387A). However, no change in phosphorylation was observed when a neighbouring serine residue
(Ser385 ) was mutated. Finally, an antibody specific to human
Ago2 phospho-Ser387 (pS387) was generated that reacts with wildtype Ago2 but not with Ago2 S387A or wild-type Ago2 that
was pre-treated with phosphatase, clearly indicating that Ago2
is phosphorylated on Ser387 . Since Ago2 is the only human Ago
protein with endonucleolytic activity, it would be very interesting
to investigate whether phosphorylation of Ser387 has functional
consequences on Ago2 cleavage activity and therefore RNAi
(RNA interference) efficiency in general. In contrast, analysis
of the phosphorylation status of other human Ago proteins may
lead to a better understanding of Ago protein function in miRNAguided gene silencing.
What are the functional consequences of Ago2 Ser387
phosphorylation? In order to address this question, Zeng et al. [9]
expressed tagged wild-type as well as the Ago2 S387A mutant in
H1299 cells and analysed its subcellular localization. Compared
with wild-type Ago2, the Ago2 S387A mutant localization to
P-bodies (processing bodies) was significantly reduced although
both proteins were expressed at similar levels. P-bodies have been
implicated in RNA metabolism as well as miRNA-guided gene
silencing [10]. The contribution of P-bodies to miRNA function,
however, remains controversial. It has been demonstrated that the
CAT1 mRNA is regulated by miR-122 in liver cells and targeted to
P-bodies for storage [11]. The CAT1 gene product is required for
an efficient cellular stress response, and upon stress induction the
1
Key words: Argonaute protein, microRNA (miRNA), phosphorylation, processing body (P-body), RNA interference (RNAi),
short interfering RNA (siRNA).
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
e8
Figure 1
S. Rüdel and G. Meister
Schematic representation of the miRNA and the siRNA pathway
For a detailed description see the main text. The illustration summarizes the miRNA and siRNA pathways. The observed differences between these pathways result mainly from the unequal
degree of complementarity between the small RNA and the targeted mRNA. hClp1 is a siRNA-specific kinase. TRBP, HIV transactivating response RNA-binding protein; PACT, PKR activator; m7 G,
7-methylguanosine cap; AAA...A, poly(A)-tail.
CAT1 mRNA is released from miRNA-guided repression and Pbody localization allowing for active translation. In contrast, it has
been demonstrated for other miRNA target mRNAs that P-bodies
are dispensable for miRNA-guided gene silencing. Therefore it
will be very interesting to analyse effects of Ago phosphorylation
on miRNA-guided gene silencing and the contribution of P-bodies
to Ago function. It is further known that Ago proteins accumulate
in cytoplasmic foci termed stress granules under cellular stress
conditions [12]. However, the Ago2 S387A mutant still localizes
to stress granules upon stress induction by arsenite treatment
indicating that Ser387 phosphorylation is not required for Ago
recruitment to stress granules.
What are the cellular pathways and, more importantly, what
is the cellular kinase that modifies Ago2 at Ser387 ? Zeng et al.
[9] addressed this question by analysing phosphorylation of
tagged Ago2 under different cellular conditions. Induction of the
cellular stress response by arsenite treatment led to a significant
increase in Ago2 Ser387 phosphorylation, suggesting that putative
Ago2 kinases are induced under stress conditions. It is well
c The Authors Journal compilation c 2008 Biochemical Society
established that the MAPK (mitogen-activated protein kinase)
pathway is activated as response to cellular stress. Indeed, a
chemical inhibitor of p38 MAPK inhibits stress-induced Ago2
phosphorylation [9]. However, recombinant p38 MAPK failed
to phosphorylate Ago2. The authors therefore speculated that
other kinases within the MAPK pathway might function on Ser387
of Ago2. Consequently, Zeng et al. [9] identified MAPKAPK2
(MAPK-activated protein kinase 2), a kinase, which is activated by
the MAPK pathway, and showed that recombinant MAPKAPK2
phosphorylates recombinant human Ago2 at Ser387 . MAPKs
are members of signalling pathways that transfer extracellular
signals from the cell membrane to the nucleus allowing the cell
to respond to extracellular stimuli. Such signalling pathways
are very often affected in various types of cancer leading to
constitutive gene activation. Furthermore, miRNAs have been
implicated in cancer pathogenesis as well. It is reasonable that Ago
phosphorylation by MAPKs might be linked to cancer biology.
It will be very interesting to investigate whether or not Ago2
Ser387 phosphorylation is inhibited or stimulated in cancer tissue
Commentary
samples. Studies on post-translational modifications of protein
factors involved in gene silencing will therefore not only elucidate
the molecular mechanisms of gene silencing but also help to
understand the molecular basis of different forms of cancer.
Our research is supported by the Max–Planck Society, the Deutsche
Forschungsgemeinschaft, the Deutsche Krebshilfe, the German ministry for Education
and Science and the European Union.
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Received 19 June 2008; accepted 23 June 2008
Published on the Internet 15 July 2008, doi:10.1042/BJ20081244
c The Authors Journal compilation c 2008 Biochemical Society