Dear Editor,
The mammalian target of rapamycin (mTOR), as a critical energy sensor and cell-growth
regulator, controls protein synthesis, autophagy and many important cellular processes
through forming functional distinct complexes, mTORC1 and mTORC2. mTORC1 that is sensitive
to rapamycin, regulates cell growth and protein synthesis, while mTORC2 that is insensitive
to rapamycin, regulates cellular metabolism and the cytoskeletal organization (Gingras
et al., 2001; Hay and Sonenberg, 2004). Translation initiation is the rate-limiting
step in protein synthesis, which proceeds through a multi-step process that can be
divided into three major steps. First, eukaryotic translation initiation factor 2
(eIF2) binds with GTP and methionyl-tRNA to form the ternary complex, which further
binds to 40S ribosomal subunit with the help of eIF1, eIF1A, eIF3 and eIF5 resulting
the preinitiation complex (PIC). Second, the PIC binds to mRNA which is unwinded by
the eIF4F complex (including eIF4E, eIF4G, eIF4A, eIF4B). Finally, the second GTPase,
eIF5B catalyzes the joining of the 60S subunit and 40S subunit to form the 80S initiation
complex (Jackson et al., 2010). mTORC1 regulates activities of a number of constituents
of protein synthesis, including translation initiation factors and elongation factors,
through phosphorylating two well-known substrates, the ribosomal S6 kinases (S6K1
and S6K2) and the eukaryotic initiation factor 4E binding proteins (4E-BP1 and 4E-BP2)
(Gingras et al., 2001; Hay and Sonenberg, 2004). Phosphorylation of 4E-BP1 by mTORC1
increases the availability of eIF4E. Phosphorylation of eIF4B by S6K1 is necessary
for PIC formation (Gingras et al., 2001). Ribosomal protein S6 (RPS6) which is the
substrate of S6K1 and S6K2, involves in the regulation of cell proliferation, cell
size and glucose homeostasis (Ruvinsky et al., 2005). However, systematic analysis
of mTORC1 mediated phosphorylation in ribosomal and associated proteins has not been
achieved.
To analyze mTORC1-mediated phosphorylation in ribosomal and associated proteins, quantitative
phosphoproteomic analysis based on the SILAC (stable isotope labeling by amino acids
in cell culture) method was carried out to analyze affinity enriched phosphoproteins
from the untreated and rapamycin-treated 293T cells. The experimental workflow was
displayed in Fig. 1A. Briefly, cells grown in light medium (12C6
14N2-Lysine and 12C6-Arginine, K0R0) were treated with 200 nmol/L rapamycin for 2
h, while cells grown in heavy medium (13C6
15N2-Lysine and 13C6-Arginine, K8R6) were untreated. Sucrose cushion centrifugation
was used to isolate ribosomes. Proteins extracted from the whole cell lysate or the
isolated ribosome fraction of the untreated and rapamycin-treated cells were mixed
and trypsin digested. Then phosphopeptides were enriched with TiO2 beads and analyzed
by nano-LC-MS/MS. The generated MS/MS spectra were searched against the human database
using the Sequest search engine in the Proteome Discoverer (Version 1.4) software,
with the false discovery rate (FDR) setting to 0.01.
Figure 1
Phosphoproteomics analysis of ribosomal proteins after rapamycin treatment. (A) A
schematic of the experimental procedure; (B) A list of mTOR-mediated phosphopeptides
from ribosomal proteins; (C) Kinases and motifs analysis of mTOR-mediated phosphorylated
peptides; (D) Locations of the mTOR-mediated phosphoresidues on human 80S ribosome.
Proteins of the 40S subunit are shown in green, proteins of the 60S subunit are shown
in blue, elongation factor 2 and guanine nucleotide-binding protein subunit beta-2-like
1 are shown in orange, and the mTOR-mediated phospho-residues are shown as red balls.
The expanded view shows the location of phosphorylation sites on RPS6, RPS8 and RPL24
We identified 61 unique phosphorylation sites on 37 ribosomal proteins, among which
33 phosphorylation sites have been reported before in the Uniprot database (http://www.uniprot.org).
Abundance of 28 phosphorylated peptides corresponding to 31 phosphorylation sites
were decreased after rapamycin-treatment (Fig. 1B), of which 12 were from 9 proteins
of the 40S ribosome, and 16 were from 12 proteins of the 60S ribosome. Quantitative
proteomics using Tandem Mass Tags (TMT) was carried out and showed that 2-h rapamycin
treatment did not induce changes in the expression levels of ribosomal proteins (Table
S1). This indicated that phosphorylation changes in these proteins were caused by
inhibition of mTOR. We further identified that 14 phosphorylated peptides possessed
three types of consensus sequence motifs using iceLogo (http://iomics.ugent.be/icelogoserver/)
(Fig. 1C and Table S2), including proline-directed motif (SP and TP), which was the
known mTOR targeted phosphorylation sites, the ribosomal S6 kinase (RSK)-targeted
arginine-rich motif (RRRxS), and the acidic motif (SxxD/E/pS) that was the potential
substrate of casein kinase II (CKII) predicted using KinasePhos (http://kinasephos.mbc.nctu.edu.tw/).
Furthermore, out of the 31 mTORC1-mediated phosphorylation sites, 20 were on the surface
of the 80S ribosome as highlighted with red color (Fig. 1D), which was downloaded
from the Protein Data Bank (accession number: 4V6X). This suggests that mTOR and associated
kinases facilely access and directly phosphorylate the 80S ribosome. Phosphorylation
sites RPS6 Ser148, Ser235, Ser236, RPS8 Ser160 and RPL24 Ser86 were on the interface
between 40S subunit and 60S subunit, indicating that mTOR mediated the interaction
of 40S and 60S subunits. Phosphorylation of the C-terminal residues in RPS6 by mTOR
is known to enhance the binding of 40S ribosome to the m7GpppG cap of mRNA for facile
translation initiation (Hutchinson et al., 2011). Rapamycin-responsive RPS8 Ser160
and RPL24 Ser86 were in close proximity to the C-terminus of RPS6, suggesting mTOR-mediated
RPS8 and RPL24 phosphorylation may also involve in mRNA binding. In summary, our phosphoproteomic
results suggest that mTOR mediated the formation of the 80S ribosome and its binding
with mRNA.
We identified 8353 unique phosphorylated peptides from 2761 distinct proteins extracted
from the whole cell lysate. Abundance of ten phosphorylation peptides corresponding
to eleven phosphorylation sites on seven translation initiation factors eIF2A, eIF3C,
eIF3E, eIF4B, eIF4G1, eIF4G2 and eIF5B were decreased after rapamycin treatment (Fig. 2A
and Table S3) and designated as mTOR regulated. eIF5B is responsible for the joining
of 60S subunits with the pre-40S subunits and plays an important role in both of the
cap-dependent and the internal ribosome entry site (IRES) dependent translation initiation
(Jackson et al., 2010; Thakor and Holcik, 2012). In the present study, phosphorylation
in four serine residues of eIF5B was decreased upon rapamycin treatment including
phosphorylation on Ser214 residue, in consistent with the early report on mTOR or
mitotic associated phosphoproteomics (Hsu et al., 2011; Kettenbach et al., 2011).
A MS/MS spectrum that matched to the fragments of the peptide (NKPGPNIEpSGNEDDDASFK)
containing phosphorylated Ser214 was shown in Fig. S1. Then we used the method of
parallel reaction monitoring (PRM) based mass spectrometry combined with tandem mass
tags (TMT) labeling to determine the intensity ratio of the phosphorylation peptide
vs. the non-phosphopeptide and confirmed that the phosphorylation ratio was decreased
from 0.3 to 0.1 upon rapamycin-treatment (Fig. S2).
Figure 2
Analysis of mTOR-mediated phosphorylation changes in eukaryotic translation initiation
factors. (A) Identified phosphorylation sites on translation initiation factors. The
mTOR-mediated phosphorylation sites are red-coded. (B) GO analysis of the binding
partners of the wild type eIF5B according to the associated biological processes;
(C) A list of proteins preferentially binding to the eIF5B-S214E mutant; (D) Reciprocal
immunoprecipitation of Nat10 and eIF5B; (E) The ratio of Ser214-containing phosphopeptide
to the nonphosphopeptide determined by PRM-based MS analysis in eIF5B from the 293T
cells and Nat10-immunoprecipiated complex. Data were analyzed using student’s t test.
***P < 0.001; n = 3
The mTOR-mediated phosphorylation in eIF2, eIF3, eIF4B and eIF4G has been reported
before (Gingras et al., 2001; Hay and Sonenberg, 2004; Ozcan et al., 2008; Martineau
et al., 2014). The present study also revealed that mTOR-inhibition decreases the
phosphorylation in eIF5B. To explore effects of Ser214 phosphorylation on eIF5B functions,
we used the CRISPR-cas9 technology to knockdown the expression of eIF5B, showing that
the expression level of eIF5B in eIF5B-knockdown cells was one third of that in the
control cells as determined by qPCR (Fig. S3). Then, plasmids containing the wildtype
eIF5B, and two mutants eIF5B-S214A and eIF5B-S214E were transiently transfected into
the eIF5B-knockdown cells, respectively, followed by immunoprecipitation to identify
the binding partners of eIF5B and mutants. The mRNA level and protein expression of
wild type eIF5B and two mutants were detected by qPCR, Western blotting and mass spectrometry,
as shown in Figs. S3, S4 and S5. The binding partners of the wild type eIF5B and two
mutants were identified by label free quantification (LFQ) using the MaxQuant software.
When a protein was only present in the eIF5B immunoprecipitated complex or the total
ion intensity of corresponding tryptic peptides from one protein identified in the
eIF5B immunoprecipitated complex is five times higher than that in the FLAG-only immunoprecipitated
complex, this protein was considered as the binding partner of eIF5B. Forty five binding
partners (Table S4) of the wild type eIF5B were identified in two independent biological
replicates and the interactome was analyzed using STRING software (http://string.embl.de)
(Fig. S6). To understand the biological relevance of these proteins, the Gene Ontology
(GO) was used to cluster proteins according to their associated biological processes.
The annotations of gene lists were summarized via a pie plot based on KEGG pathway
analysis as shown in Fig. 2B. Eighty percent of eIF5B-binding proteins participated
in the genetic information processing, including ribosome biogenesis, messenger RNA
biogenesis and spliceosome. In comparing to proteins binding to the wild type eIF5B,
several proteins were found to preferentially bind to eIF5B-S214E mutant, but not
eIF5B-S214A mutant as displayed in Fig. 2C. These proteins were considered to have
the higher affinity to Ser214-phosphorylated eIF5B. Among them, N-acetyltransferase
10 (Nat10), a RNA acetyltransferase, is responsible for 18S rRNA processing through
inducing N4-acetylcytidine formation (Ito et al., 2014). The Nat10-eIF5B interaction
was verified by reciprocal immunoprecipitation (Fig. 2D) and eIF5B was immunoprecipitated
by Nat10-specific antibody in Nat10-transfected 293T cells. Using PRM-based targeted
mass spectrometry, we found that the ratio of the phosphorylated Ser214-containing
peptide to the unphosphorylated peptide in the Nat10-immunoprecipitated eIF5B was
3 times higher than that in eIF5B from 293T cells, which confirmed that Nat10 preferred
to bind to Ser214 phosphorylated eIF5B (Fig. 2E). It was reported that the mTOR pathway
involved in rRNA maturation with unknown mechanism (Iadevaia et al., 2012). Our results
suggested that mTOR phosphorylated eIF5B to enhance the binding of eIF5B with Nat10
and to promote rRNA processing.
Taken together, we applied quantitative phosphoproteomics to profile rapamycin-inhibition
mediated phosphorylation changes in ribosomal proteins and suggested that mTOR mediated
the 80S ribosome formation and its binding to mRNA. We further mapped mTOR-regulated
phosphorylation sites in eukaryotic translation initiation factors and identified
that phosphorylation of Ser214 on eIF5B enhanced the binding of eIF5B to the RNA-binding
and processing proteins including Nat10. Our results suggest that the interaction
of phosphorylated eIF5B with Nat10 promotes rRNA processing and rRNA maturation.
FOOTNOTES
We thank the Protein Chemistry Facility at the Center for Biomedical Analysis of Tsinghua
University for sample analysis. This work was supported in part by the National Natural
Science Foundation of China (Grant No. 31270871 to H.T.D) and MOEC 2012Z02293 (H.T.D),
the National Basic Research Program (973 Program) (No. 2014CBA02005 to H.T.D.) and
the Global Science Alliance Program of Thermo-Fisher Scientific.
Xu Jiang, Shan Feng, Yuling Chen, Yun Feng and Haiteng Deng declare that they have
no conflict of interest. This article does not contain any studies with human or animal
subjects performed by the any of the authors.
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