From the Department of Human Biological Chemistry and
Genetics,
Department of Pharmacology and Toxicology, the
§ Sealy Center for Cancer Cell Biology, University of Texas
Medical Branch, Galveston, Texas 77555 and the ¶ University of
Rochester School of Medicine and Dentistry, Department of Biochemistry
and Biophysics, Rochester, New York 14642
Received for publication, March 23, 2001, and in revised form, April 19, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have cloned and characterized a new member of
the phosphatidylinositol kinase (PIK)-related kinase family. This gene,
which we term human SMG-1 (hSMG-1), is
orthologous to Caenorhabditis elegans SMG-1, a protein that
functions in nonsense-mediated mRNA decay (NMD). cDNA
sequencing revealed that hSMG-1 encodes a protein of
3031 amino acids containing a conserved kinase domain, a C-terminal domain unique to the PIK-related kinases and an FKBP12-rapamycin binding-like domain similar to that found in the PIK-related
kinase mTOR. Immunopurified FLAG-tagged hSMG-1 exhibits protein kinase activity as measured by autophosphorylation and phosphorylation of the
generic PIK-related kinase substrate PHAS-1. hSMG-1 kinase activity is
inhibited by high nanomolar concentrations of wortmannin (IC50 = 105 nM) but is not inhibited by a
FKBP12-rapamycin complex. Mutation of conserved residues within the
kinase domain of hSMG-1 abolishes both autophosphorylation and
substrate phosphorylation, demonstrating that hSMG-1 exhibits intrinsic
protein kinase activity. hSMG-1 phosphorylates purified hUpf1 protein,
a phosphoprotein that plays a critical role in NMD, at sites that are
also phosphorylated in whole cells. Based on these data, we conclude
that hSMG-1 is the human orthologue to C. elegans
SMG-1. Our data indicate that hSMG-1 may function in NMD by
directly phosphorylating hUpf1 protein at physiologically relevant sites.
The PIK1-related kinases are a subfamily of the
phosphatidylinositol (PI) kinases based
on homology to the core catalytic domain of phosphatidylinositol
3-kinase (PI3K) (1, 2). Regions of homology between PI kinases and the
protein kinase superfamily include the ATP-binding site and the
catalytic/substrate-binding site (3). PIK-related kinases are distinct
from PI kinases in that they are high molecular weight proteins that
function as serine/threonine protein kinases, rather than lipid kinases (3). PIK-related kinases can be divided into three subgroups based on
structural and functional similarities shared by certain family
members. The ATM/ATR/RAD3 subgroup functions in DNA damage response
pathways, and members contain regions called RAD3 homology domains (4,
5). The targets of rapamycin or TORs (Saccharomyces cerevisiae TOR1 and TOR2 and human mTOR/FRAP/RAFT1/RAPT1) were originally identified as intracellular targets of the
immunophilin-immunosuppressant complex FKBP12-rapamycin (6, 7). TORs
share sequence similarity within the FRB domain, which binds this
complex (7). The TORs function in response to mitogenic signaling to
regulate cap-dependent translation (8). Finally, the
catalytic subunit of DNA-dependent protein kinase
(DNA-PKcs) shares no sequence similarity to other PIK-related kinases, aside from the kinase domain. DNA-PK
functions in the repair of programmed DNA breaks generated by meiotic
and V(D)J recombination, and those generated by genotoxic insults (9).
Recently, evidence has emerged for an essential PIK-related kinase in
nonsense-mediated mRNA decay (NMD). NMD, or mRNA surveillance, is an evolutionarily conserved process by which mRNA species
containing premature termination codons are preferentially degraded,
thereby preventing accumulation of truncated proteins that might serve in a dominant negative or gain-of-function manner (11-18). NMD also
functions to regulate the level of a number of normal mRNAs (19-22). NMD has been genetically analyzed in yeast and nematodes. Seven genes (SMG-1 to SMG-7) are involved in NMD
in Caenorhabditis elegans (23). One of these genes,
SMG-1 (ceSMG-1), is predicted by sequence to
encode a PIK-related kinase (10). Biochemical studies of ceSMG-1 are
lacking, and the protein has never been shown to have
phosphotransferase activity. However, genetic evidence indicates that
phosphorylation of SMG-2, an RNA helicase, is blocked in
ceSMG-1 mutants (10). SMG-2 and its human homologue, human Upf1 (hUpf1), contain multiple potential PIK-related kinase (S/T)-Q and
(S/T)-P phosphorylation site motifs (24). Furthermore, phosphorylation of hUpf1 is blocked by high concentrations of wortmannin
(IC50 = 100 nM), consistent with the role of a
PIK-related kinase (24). Such observations suggest that ceSMG-1 is a
PIK-related kinase that may phosphorylate SMG-2. However, direct
phosphorylation of SMG-2 or hUpf1 protein by ceSMG-1 or an SMG-1-like
kinase in mammalian cells has never been demonstrated.
In the present report, we describe the cloning and characterization of
the human orthologue to ceSMG-1, which we term human SMG-1 (hSMG-1). hSMG-1 encodes a novel
PIK-related kinase with significant homology to the TORs and ceSMG-1.
Biochemical characterization of transiently expressed FLAG-tagged
hSMG-1 protein indicates that it is a protein kinase that exhibits both
autophosphorylation and substrate-specific phosphorylation. FLAG-hSMG-1
directly phosphorylates hUpf1 protein at sites phosphorylated in whole
cells, indicating that hSMG-1 is a physiologically relevant hUpf1
protein kinase.
hSMG-1 cDNA Cloning and Plasmid Construction--
5'-RACE
(rapid amplification of cDNA
ends) was performed using K562 (human chronic myelogenous
leukemia) Marathon Ready cDNA (CLONTECH
Laboratories, Inc.) and gene-specific primers toward the 5'-end of
KIAA0421 (NCBI gi: 2887416; accession number
AB007881), a partial cDNA with significant sequence similarity to
PIK-related kinases, to generate an additional 1000 base pairs of
cDNA sequence. The remaining hSMG-1 cDNA sequence
was obtained from a human prostate 5'-STRETCH (
FLAG-tagged, full-length hSMG-1 cDNA was generated by
PCR amplification of three overlapping hSMG-1 fragments
corresponding to nucleotides 1-2580 (fragment A), nucleotides
2531-6540 (fragment B), and nucleotides 6481-9096 (fragment C). The
initiating methionine codon was mutated to glycine (ATG to GTG) to
generate a 5'-FLAG-tagged cDNA. The A, B, and C fragments were
cloned into pCR®-XL-TOPO (Invitrogen), and FLAG-tagged,
full-length cDNA (pCMV-FLAG-hSMG-1) was assembled by directional
cloning into the pCMV-TAG2B (Stratagene) and then subcloned into
pCI-neo (Promega). Clones were verified by sequencing.
Three kinase-deficient (KD) hSMG-1 cDNAs were generated
by site-directed mutagenesis at nucleotide 5114 (GAC to GCC), or
nucleotide 5171 (GAT to GAG or GAT to GCT) within motif I (KD1) and
motif II (KD2 and -3) of the catalytic domain. All hSMG-1
clones DNA were confirmed by sequencing. Sequence alignments were
performed using the Vector NTI Suite II program (InforMax Inc.).
Northern Blot Analysis--
Total cellular RNA from human HL60,
K562, and HEK-293 cells was extracted using TRIzol®
reagent (Life Technologies, Inc.). Equal amounts (5 µg) of RNA was
resolved in formaldehyde-agarose gels, transferred to nitrocellulose, and hybridized with one of several probes spanning hSMG-1 as
indicated in the legend to Fig. 2. The expression of hSMG-1 mRNA in
the Human Tissue 12-Lane MTN® Blot and Human Cancer Cell
Line MTN® Blot (CLONTECH Laboratories,
Inc.) was assessed by Northern blot hybridization. hSMG-1 Polyclonal Antibody Production and Immunoblot
Analysis--
A polyclonal rabbit antiserum was raised (Alpha
Diagnostics International, San Antonio, TX) against GST-hSMG-1 fusion
protein consisting of the C-terminal 419 amino acids of hSMG-1. PCR
product from a reaction using the following primers (5'-GAA GAA TTC ATT GCG ACA GTT CAG GAG AAG-3' and 5'-CTG GCG GCC GCC TCA CAC CCA GGC
TGT-3') was cloned into pCR®2.1-TOPO (TOPO TA
Cloning® Kit, Invitrogen). The PCR product was then cloned
into the EcoRI and NotI sites of pGEX-4T-1
(Amersham Pharmacia Biotech). GST-hSMG-1 was expressed in E. coli, and cell pellets from
isopropyl-
Affinity-purified antibody was generated using purified GST-hSMG-1
resolved by SDS-PAGE in 10% acrylamide preparative gels, transferred
to nitrocellulose, and visualized by staining with fast green. The area
of nitrocellulose containing GST-hSMG-1 was excised, incubated with 1%
bovine serum albumin in PBS containing 0.1% Tween 20 (1% BSA/PBST),
and then incubated with a 1:1000 dilution of anti-hSMG-1 serum in 1%
BSA/PBST for 1 h at room temperature. The nitrocellulose was
washed twice for 10 min in PBST, once in PBS alone, and antibody eluted
into a minimal volume of 0.5 M acetic acid at pH 2.5 for 2 min. Eluted antibody was immediately neutralized by addition of 3 volumes of 1% BSA/PBS and dialyzed three times for 30 min against
ice-cold PBS. Purified antibody was concentrated using a Centricon-10
(Amicon Inc.) and stored at
Immunoblot analysis of hSMG-1 was performed using a 1:10 dilution of
affinity-purified antibody in 1% BSA/PBST. Total cell lysates from
HEK-293 cells were separated in a 7.5% acrylamide Tris-HCl gel
(Bio-Rad), transferred to nitrocellulose and blocked in 5% nonfat dry
milk/PBST for 1 h prior to incubation with affinity-purified hSMG-1 antibody for 2 h. Membrane was washed three times in PBST, incubated in a 1:10,000 dilution of goat anti-rabbit IgG (Kirkegaard & Perry Laboratories) in 5% milk/PBST for 1 h, and washed two times
in PBST and once in PBS. Chemiluminescent detection of antigen-antibody complexes was performed using SuperSignal® West Pico
substrate (Pierce).
Cell Lines and Transfections--
Human chronic myelogenous
leukemia K562 cells and promyelocytic leukemia HL60 cells were
maintained in suspension culture in Iscove's medium (Life
Technologies, Inc.) supplemented with 10% bovine calf serum (K562
cells) or 10% fetal calf serum (HL60 cells), 100 µg/ml penicillin,
and 100 IU/ml streptomycin as described previously (25). Human
embryonic kidney 293 (HEK-293) cells were maintained in DMEM medium
(Life technologies, Inc.), 10% bovine calf serum, 100 µg/ml
penicillin, and 100 IU/ml streptomycin. Subconfluent HEK-293 cells were
transiently transfected using LipofectAMINE Plus (Life Technologies,
Inc.) with ~1-3 µg of DNA/plate and harvested for analysis 48 h after transfection.
hSMG-1 Immunopurification and Protein Kinase Assay--
FLAG-WT-
or -KD-hSMG-1 was immunopurified from transiently transfected HEK-293
cells using anti-FLAG M2® conjugated agarose beads
(Sigma). Briefly, cells were rinsed once with ice-cold 1× PBS,
scraped, and transferred into microcentrifuge tubes, and lysed
in 200 µl of lysis buffer (50 mM
Protein kinase assays were performed in kinase buffer containing 100 µM ATP, 10 mM MnCl2, 1 µg of
recombinant PHAS-1 (Calbiochem) or immunopurified FLAG-hUpf1 protein,
and 5 µCi of [
As a positive control for rapamycin inhibition, mTOR was immunopurified
from HEK-293 cells using mTAb1 (27). Briefly, 1000 µg of clarified
cell lysate was incubated with 5 µg of mTAb1 antibody conjugated to
20 µl of protein A-Sepharose 4B beads (Sigma) for 2 h at
4 °C. Beads were washed as described above and treated with or
without 10 µM rapamycin and 10 µg of FKBP12 for 1 h. After washing twice with kinase buffer, kinase activity was assessed using 1 µg of PHAS-1 as substrate as described above. Immunoblot analysis of immunopurified mTOR was performed using 1:1000 dilution of
mTAb1 antibody in 5% nonfat dry milk/PBST for 1 h at room
temperature. Membranes were washed three times in PBST, incubated with
a 1:10,000 dilution of goat anti-rabbit IgG (Kirkegaard & Perry
Laboratories) in 5% nonfat dry milk/PBST for 1 h, washed, and
subjected to chemiluminescent detection.
Comparative Two-dimensional Tryptic Phosphopeptide Analysis of
hUpf-1--
32P-Labeled FLAG-hUpf-1 protein was obtained
from HEK-293 cells transiently transfected with pCI-FLAG-hUpf-1 for
24 h. Cells were washed twice with DMEM without phosphate (Life
Technologies, Inc.) and incubated with 100 µCi/ml of
[32P]orthophosphate (ICN Pharmaceuticals) in DMEM
supplemented with 5% bovine calf serum for 12 h. Cells were
harvested, lysed, and FLAG-hUpf1 protein immunopurified as described
above. 32P-Labeled FLAG-hUpf1 protein phosphorylated
in vitro by immunopurified FLAG-hSMG-1 was generated as
described above. Samples were analyzed in 3-8% acrylamide NuPAGE Tris
acetate gels (Invitrogen) and transferred to nitrocellulose.
Radioactivity was visualized by InstantImager, and
32P-labeled hUpf-1 protein was excised from the
nitrocellulose, rinsed six times with dH2O, three times
with 50 mM NH4HCO3, and digested
overnight with 1 mg/ml trypsin in 100 µl of 50 mM
NH4HCO3. Fresh trypsin (1 mg/ml) was added
after 18 h and digestion continued for an additional hour. Digests
were lyophilized in a speed-vac, washed several times with
dH2O, and relyophilized. Two-dimensional phosphopeptide
mapping was performed as described previously (28, 29). Electrophoresis
was performed in pH 1.9 buffer (formic acid/acetic
acid/dH2O; 50:156:1794), and chromatography was performed in isobutyric chromatography buffer (isobutyric
acid/n-butanol/pyridine/glacial acetic
acid/dH2O, 65:2:5:3:25). Radioactivity was visualized by InstantImager and autoradiography on X-Omat film.
Cloning of hSMG-1 cDNA--
In a search for sequences with
homology to ceSMG-1, we used the GenBankTM data
base of ESTs to identify a partial cDNA, termed KIAA0421 (NCBI gi: 2887416; accession number AB007881) with homology to
PIK-related kinases (30). Both 5'-RACE and PCR screening were used to
clone the full-length hSMG-1 cDNA as described under "Experimental Procedures." The starting methionine codon was
identified using two criteria: 1) the presence of upstream in-frame
stop codons in three independent clones and 2) a Kozak consensus
context. The open reading frame is encoded by 9096 base pairs and
predicts a protein of 3031 amino acids with a molecular mass of 340,674 daltons and a pI of 6.00. The full nucleotide and amino acid sequence of hSMG-1 can be accessed through GenBankTM
accession number AY014957 (NCBI gi: 372334). Interestingly, a
fourth independent clone contained a unique sequence upstream of the
designated start codon, which extended the open reading frame,
suggesting that alternative splicing variant(s) of hSMG-1 may exist.
hSMG-1 Shares Sequence Homology with Members of the PIK-related
Kinase Family--
Comparison of the deduced amino acid sequence of
hSMG-1 with the protein data base revealed high sequence
homology with the PIK-related kinase family of protein kinases,
including 1) the ataxia telangiectasia gene product, ATM, and the
related human ATR and yeast RAD3; 2) the targets of rapamycin (yeast
TOR1, TOR 2, and the human homologue mTOR/FRAP/RAFT1/RAPT1); and 3) the catalytic subunit of DNA-dependent protein kinase
(DNA-PKcs). hSMG-1 contains several highly conserved motifs
found in all PIK-related kinases, including a conserved ATP-binding
site (Lys1525), and motif I
(D1705XXXXN1710) and motif II
(D1724XX) sites within the catalytic domain
(Fig. 1C). In addition, hSMG-1
contains a short C-terminal region at amino acids 3001-3031 (termed
FATC for FRAP, ATM, TRRAP at
C-terminus), found in the majority of the PIK-related
kinases (Fig. 1A) (31). Among the well characterized
PIK-related kinases, hSMG-1 exhibits the highest sequence homology to
the TORs, which extends beyond the kinase domain to the FRB
(FKBP12-rapamycin binding) domain (Fig. 1, A and
B). Mutational analysis of the FRB domain of mTOR has
defined it as the site of binding for rapamycin, a selective inhibitor of TOR (8, 32). Several residues within this domain are also required
for mTOR kinase activity (8). Sequence alignment indicates that hSMG-1
lacks a critical serine residue that is required for binding of the
FKBP12-rapamycin complex to mTOR (Fig. 1B) (32). However,
the FRB domain of hSMG-1 retains a conserved tryptophan residue, which
when mutated in mTOR abolishes kinase activity (Fig. 1B)
(8). Conservation of this tryptophan residue suggests that it is
important for hSMG-1 kinase activity.
The overall structure of hSMG-1 differs from that of other PIK-related
kinases, since the kinase domain is not located at the extreme C
terminus (Fig. 1A). Rather, a large region is located between the kinase domain and the FATC region that is 100% identical to LIP (lambda-interacting protein). LIP was identified by yeast two-hybrid screening as a protein that interacts with the zinc finger
domain of PKC hSMG-1 Shares Sequence Homology to ceSMG-1, a C. elegans Protein
Required for Nonsense-mediated mRNA Decay--
In addition to
homology to mTOR, hSMG-1 exhibits significant homology to ceSMG-1 in
three major regions: an N-terminal ~200-amino acid region of 44%
similarity, a ~1000-amino acid region in the middle of the coding
sequence of 46% similarity that includes the FRB-like and kinase
domains, and a C-terminal ~88-amino acid region of 61% similarity
encompassing the FATC domain (Fig. 1A). Two additional
regions of homology between ceSMG-1 and hSMG-1 (termed SMG-1 homology
Domains 1 and 2: SD1 and SD2) have not previously been described in
other PIK-related kinases and may constitute unique functional domains
characteristic of ceSMG-1 and hSMG-1.
hSMG-1 Is Widely Expressed as a High Molecular Weight RNA and
Protein--
To assess hSMG-1 expression, RNA blot hybridization was
performed using total RNA from K562, HL60, and HEK-293 cells (Fig. 2, A-C). hSMG-1 is expressed
as an RNA of ~11.6 kb in HL60 and K562 cells as determined using a
probe spanning nucleotides 5937-6890 (probe 1) of hSMG-1 (Fig.
2A). This probe overlaps with the first 150 nucleotides of
the reported LIP sequence, suggesting that the reported ~7.5-kb LIP
mRNA (33) is not expressed in these cells. To confirm this
observation, RNA blot hybridization was performed using a LIP-specific
probe (probe 2, nucleotides 6741-7812) and a hSMG-1-specific probe
(probe 3, nucleotides 3521-4654). Like probe 1, these two probes
detect an ~11.6-kb RNA in HL60, K562, and HEK-293 cells, but no
smaller RNA(s) corresponding to LIP (Fig. 2, B and
C). RNA blot hybridization of cancer cell lines and human
tissues using probe 1 (Fig. 2, D and E) detected
hSMG-1 RNA in each of the cancer cell lines with relatively low levels in lung carcinoma (A459) and melanoma (G361) cell lines. hSMG-1 RNA was
also detected in the majority of human tissues at varying levels.
Therefore, hSMG-1 RNA is widely expressed in multiple tissues and cell
lines.
Immunoblot analysis using an affinity-purified hSMG-1 antibody detected
a single immunoreactive band in HEK-293 cells with an apparent
molecular mass consistent with the predicted molecular mass of 340 kDa.
Importantly, no lower molecular mass bands of ~80 kDa
consistent with LIP (33) were detected. The antigen used to produce our
hSMG-1 antibody consisted of the C-terminal region of the reported LIP
cDNA. Therefore, it is likely that our antibody would detect LIP if
it were expressed in these cells. We have also failed to detect LIP by
immunoblot analysis in K562 and HL60 cells (data not shown).
hSMG-1 Is a Bona Fide PIK-related Kinase--
To assess hSMG-1
protein kinase activity, full-length wild-type (WT) and
kinase-deficient (KD1) hSMG-1 were expressed as FLAG-tagged proteins in
HEK-293 cells. Cultures were transfected with empty vector (pCI-neo),
pCI-FLAG-WT-hSMG-1, or pCI-FLAG-KD-hSMG-1 and FLAG-tagged protein
immunopurified using anti-FLAG M2® beads. FLAG-hSMG-1
immunoprecipitates were assayed for autophosphorylation and
phosphorylation of recombinant PHAS-1 in the presence of
Mn2+ and [32P]ATP (Fig.
3B). PHAS-1 was chosen as
substrate because it contains multiple (S/T)-Q and (S/T)-P
phosphorylation motifs that serve as general phospho-acceptor sites for
many PIK-related kinases, including mTOR, DNA-PK, and ATM (34).
FLAG-WT-hSMG-1 phosphorylates both itself and PHAS-1, whereas
phosphorylation is not observed in immunopurified FLAG-KD-hSMG-1 above
the background level observed in FLAG immunoprecipitate from cells
transfected with pCI-neo. Immunoblot analysis confirmed the presence of
FLAG-WT-hSMG-1 and FLAG-KD1-hSMG-1 in the immunoprecipitates. Two other
FLAG-KD-hSMG-1 variants (D1724A; KD2) and (D1724E, KD3) also lacked
intrinsic kinase activity (data not shown). These single amino acid
substitutions were selected because analogous changes abolish the
protein kinase activity of other PIK-related kinases (35, 36). These
data demonstrate that hSMG-1 exhibits intrinsic protein kinase
activity. Like other PIK-related kinases, hSMG-1 kinase activity
exhibits a strong preference for Mn2+ over Mg2+
(data not shown).
An identifying feature of PIK-related kinases is sensitivity to
wortmannin at high nanomolar concentrations (IC50
~50-500 nM) (34, 37), but not at low nanomolar
concentrations that inhibit PI3K (IC50 = 5 nM)
(38). Incubation of immunopurified FLAG-WT-hSMG-1 with increasing
concentrations of wortmannin leads to dose-dependent
inhibition of both autophosphorylation and PHAS-1 phosphorylation with
an IC50 of 105 nM (Fig. 3, C and
D). hSMG-1 is also inhibited by LY294002 at low micromolar
concentrations comparable with LY294002 inhibition of PI3 kinase and
other PIK-related kinases (data not shown) (37).
The finding that hSMG-1 contains an FRB-like domain was of interest,
since to date the FRB domain has been a unique feature of the TORs
(39). Mutational analysis identified a critical serine residue within
the FRB domain of mTOR (Ser2035) that is required for
binding of FKBP12-rapamycin complex and mTOR kinase inhibition (8, 32).
Multiple sequence alignments of hSMG-1, ceSMG-1, and mTOR indicate that
hSMG-1 and ceSMG-1 lack this critical Ser residue (Fig. 1B).
To test whether hSMG-1 kinase activity is inhibited by rapamycin,
immunopurified FLAG-WT-hSMG-1 was incubated with either rapamycin
alone, rapamycin and FKBP12, FK506 alone, or FK506 and FKBP12 prior to
assay for autophosphorylation and phosphorylation of PHAS-1 (Fig.
3E). hSMG-1 kinase activity was not affected by any of the
combinations of rapamycin, FKBP12, or FK506. In contrast, mTOR
exhibited the expected inhibition by FKBP12-rapamycin complex (27, 40).
Treatment of pCI-FLAG-WT-hSMG-1 HEK-293 cell transfectants with
rapamycin prior to isolation failed to inhibit immunopurified
FLAG-hSMG-1 kinase activity (data not shown).
Our structural and biochemical analyses demonstrate that hSMG-1 is a
bona fide PIK-related kinase. Like other PIK-related kinases, hSMG-1 is a high molecular weight protein with a conserved kinase domain closely related to that of the
phosphatidylinositol-kinases. Like other PIK-related kinases, hSMG-1
phosphorylates itself and the generic PIK-related kinase substrate,
PHAS-1. hSMG-1 kinase activity is inhibited by wortmannin and LY294002
at concentrations that inhibit other PIK-related kinases. However,
hSMG-1 does not exhibit sensitivity to rapamycin inhibition despite the
presence of a FRB-like domain. The role of the FRB-like domain in
hSMG-1 function remains to be determined.
hSMG-1 Phosphorylates hUpf1 Protein at Sites Phosphorylated in
Whole Cells--
hUpf1, the human orthologue to C. elegans SMG-2
and the S. cerevisiae Upf1 protein, is an
ATP-dependent helicase required for NMD in mammalian cells
(10, 26, 41, 42). hUpf1, like SMG-2, is a phosphoprotein whose
phosphorylation is inhibited by high concentrations of wortmannin
(IC50 = 100 nM) (24). These observations, and
the fact that hUpf1 is rich in (S/T)-Q and (S/T)-P motifs that serve as
phosphorylation sites for PIK-related kinases, suggest that a
PIK-related kinase may mediate hUpf1 protein phosphorylation (24). This
hypothesis is supported by the observation that phosphorylated SMG-2 is
not detected in a ceSMG-1 mutant background (10). These biochemical and
genetic data suggest that hSMG-1 may be a physiologic hUpf1 protein kinase.
To directly test this hypothesis, immunopurified FLAG-hUpf1 protein was
used as a substrate for immunopurified FLAG-WT-hSMG-1 or FLAG-KD-hSMG-1
(Fig. 4A). FLAG-WT-hSMG-1
directly phosphorylates hUpf1 protein in vitro, whereas
FLAG-KD-hSMG-1 is incapable of doing so. To determine whether hSMG-1
phosphorylates physiologically relevant sites on hUpf1, the sites of
hSMG-1-mediated phosphorylation were compared with those phosphorylated
in whole cells. HEK-293 cells were transiently transfected with
pCI-FLAG-hUpf1 and incubated with [32P]orthophosphate for
12 h, and 32P-labeled FLAG-hUpf1 was
immunopurified. Comparative two-dimensional tryptic phosphopeptide
mapping (Fig. 4B) demonstrates that hUpf1 phosphorylated by
hSMG-1 contains two major phosphopeptides (Fig. 4B, in
vitro, labeled 1 and 2). A similar
phosphopeptide pattern consisting of two major phosphopeptides was
observed with hUpf1 phosphorylated in whole cells (Fig. 4B, whole
cells, labeled 1 and 2). Analysis of a
mixture of these digests revealed that phosphopeptides 1 and 2 are
identical. Thus, hSMG-1 phosphorylates hUpf1 at the same two major
sites phosphorylated in whole cells.
We conclude that hSMG-1 is a physiologically relevant hUPF1 kinase. The
physiologic role of hSMG-1-mediated phosphorylation of hUpf1 is
unknown. One possible role is to regulate hUpf1 function in NMD. We
have been unable to generate direct evidence of a critical role for
hSMG-1 in NMD, likely due to the low levels of KD-hSMG-1 expression
that we can achieve in transiently transfected cells (data not shown).
It is unclear whether this low level of expression is due to a
cytotoxic effect of KD-hSMG-1. Future studies aim to directly assess
the importance of hSMG-1 and hSMG-1-mediated phosphorylation of hUpf1
protein in NMD in human cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-gt10) cDNA
library (CLONTECH Laboratories, Inc.) by PCR. The
full-length cDNA sequence, derived from 12 overlapping clones, was
verified by alignment of four independent full-length clones.
Comparison of the sequence to the GenBankTM EST data base
identified KIAA0220 (NCBI gi: 1504021; accession number D86974), which corresponds to amino acids 95-647 of the
full-length hSMG-1 cDNA. The hSMG-1 coding
sequence can be accessed via GenBankTM accession number
AY014957 (NCBI gi: 372334).
-Actin mRNA
served as a control for RNA loading using human
-actin cDNA as
probe (CLONTECH Laboratories Inc.). Probes were
32P-labeled by random priming using
[
-32P]dCTP and the Megaprimer DNA labeling kit
(Amersham Pharmacia Biotech).
-D-thiogalactopyranoside-induced cultures were lysed at 4 °C in lysis buffer (100 mM
Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1.5%
Sarkosyl, 200 mg/ml lysozyme, 5 mM DTT, and protease
inhibitors) by sonication. Clarified cell lysates were supplemented
with 1% Triton X-100, and GST-hSMG-1 was bound to
glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) for 2 h at 4 °C. Affinity-purified GST-hSMG-1 was resolved in 10%
acrylamide SDS-PAGE preparative gels, visualized with 0.1 M
ice-cold KCl, excised, recovered by electroelution, and used to
immunize rabbits for polyclonal antibody production.
20 °C in PBS containing 10% glycerol.
-glycerophosphate, 10 mM potassium phosphate, 50 mM NaF, 1 mM EDTA, and 1 mM EGTA, pH 7.4) supplemented
with 1 mM DTT, 10 µg/ml each of leupeptin, pepstatin,
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM microcystin LR, 2.5 mM MgCl2,
and 0.1% Tween 20. Cells were homogenized using a 1.5-ml pestle
followed by microcentrifugation at 12,000 rpm for 20 min at 4 °C.
Immunopurification was performed by incubating 800-1000 µg of
clarified lysate with 30 µl of anti-FLAG M2®-conjugated
agarose beads for 2 h at 4 °C with gentle rotation. Beads were
washed twice with 1 ml of each of the following buffers: lysis buffer
supplemented with 1 mM DTT and 0.1% Tween 20, lysis buffer
containing 500 mM NaCl supplemented with 1 mM
DTT and 0.1% Tween 20, wash buffer (50 mM Tris, 1 mM EDTA, and 1 mM EGTA), and kinase buffer (10 mM HEPES, 50 mM NaCl, 0.1 mM EGTA,
50 mM
-glycerophosphate, 500 nM microcystin
LR, and 1 mM DTT). FLAG-hUpf1 protein was immunopurified
from HEK-293 cells transiently transfected with 1 µg of
pCI-FLAG-hUpf-1 DNA (26) for 48 h and used as substrate for
immunopurified FLAG-hSMG-1 kinase assay as described below.
-32P]ATP (PerkinElmer Life Sciences)
in a final volume of 20 µl. Reactions were performed at 30 °C and
terminated after 30 min by the addition of 20 µl of 2× SDS-PAGE
sample buffer followed by boiling for 5 min. Samples were analyzed in
4-20% acrylamide Tris-HCl (Bio-Rad) or NuPAGE 3-8% acrylamide Tris
acetate gels (Invitrogen) and transferred to nitrocellulose.
Radioactivity was detected using an InstantImager (Packard) and
autoradiography on X-Omat film (Eastman Kodak Co.). For wortmannin
treatment, immunopurified FLAG-hSMG-1 was incubated with the indicated
amount of wortmannin or solvent (Me2SO) in a final
volume of 30 µl at room temperature for 1 h, washed twice with
kinase buffer, and the kinase assay performed as indicated above. For
rapamycin treatment, immunopurified FLAG-hSMG-1 was incubated with 10 µM rapamycin or 100 µM FK506 in the
presence or absence of 10 µg GST-FKBP12 in a final volume of 50 µl.
After 1 h, beads were washed twice with kinase buffer, and kinase
assay performed as indicated above. Immunoblot analysis of FLAG-tagged
proteins was performed using anti-FLAG M2® monoclonal
antibody peroxidase conjugate (Sigma) at a 1:1000 dilution in PBST
followed by chemiluminescent detection.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (37K):
[in a new window]
Fig. 1.
Sequence of human SMG-1 and alignment to
PIK-related kinase family members. A, a
schematic diagram showing homology between hSMG-1, ceSMG-1, and mTOR.
The FRB domain (FRB), kinase domain (Kinase
Domain), and the C-terminal FATC region (FATC) are
indicated. In addition, two regions of homology between hSMG-1 and
ceSMG-1, SD1 and SD2, are indicated. Numbers
represent percent identity and similarity to hSMG-1, respectively.
B, alignment of the FRB-like domain of hSMG-1 with ceSMG-1
and mTOR. Shading indicates identical and similar amino
acids. The critical tryptophan residue required for mTOR kinase
activity is indicated (box labeled Kinase
Activity). Also indicated is a Ser residue important for rapamycin
binding to mTOR (box labeled RAP:FKBP12 site).
C, alignment of the kinase domains of hSMG-1, ceSMG-1, and
mTOR. Shading indicates identical and similar amino acids.
Indicated is the conserved Lys within the ATP binding domain
(box labeled Lys 1525), and conserved motif I and
motif II sequences (box labeled Motif I and
Motif II, respectively). Amino acid mutations introduced in
kinase-deficient hSMG-1 variants (KD#1, KD#2, and
KD#3) are indicated with arrows.
/
(33). LIP cDNA was reported to be 2142 base
pairs, coding for a protein of 713 amino acids with a predicted molecular mass of 79.7 kDa (NCBI gi: 5542015; accession number U32581) (33). The observation that LIP forms part of hSMG-1 raised the
possibility that LIP is a splicing variant of hSMG-1. However, our
Northern and immunoblot analyses argue against this hypothesis (see below).
View larger version (77K):
[in a new window]
Fig. 2.
Expression of hSMG-1 RNA. Total RNA from
HL60 (A-C), K562 (A-C), and HEK-293
(B and C) cells was hybridized with
hSMG-1-specific probes corresponding to nucleotides 5937-6890 (probe
1) (A), nucleotides 6741-7812 (probe 2) (B), or
nucleotides 3521-4654 (probe 3) (C) within the hSMG-1 cDNA. RNA
hybridization of the Human Cancer Cell Line MTN® Blot
(D) and Human 12-Lane MTN® Blot (E)
(CLONTECH Laboratories, Inc.) using probe 1. Hybridization to human -actin probe is shown as a control for RNA
loading (
-actin).
View larger version (41K):
[in a new window]
Fig. 3.
hSMG-1 is expressed in HEK-293 cells and
exhibits intrinsic protein kinase activity. A,
endogenous hSMG-1 protein expression was determined in HEK-293 cell
lysates by immunoblot analysis using affinity-purified hSMG-1 antibody.
A single band is detected with an apparent molecular mass consistent
with the predicted molecular mass of 340 kDa. B, HEK-293
cells were transfected with empty vector (pCI-neo) ( ),
pCI-FLAG-WT-hSMG-1 (WT), or pCI-FLAG-KD1-hSMG-1
(KD) and immunopurified FLAG-hSMG-1 was assayed for
autophosphorylation and phosphorylation of PHAS-1. Immunoblot analysis
using FLAG antibody (anti-FLAG) demonstrates the presence of
equivalent amounts of FLAG-WT-hSMG-1 and FLAG-KD-hSMG-1. C,
HEK-293 cells were transfected with empty vector (pCI-neo) (
),
pCI-FLAG-WT-hSMG-1 (WT), or pCI-KD-hSMG-1 (KD)
and immunopurified hSMG-1 was incubated with the indicated
concentration of wortmannin (WM (nM)) or diluent (0.1%
Me2SO) (
) for 1 h prior to assay for
autophosphorylation ([32P]hSMG-1) and
phosphorylation of PHAS-1 ([32P]PHAS-1).
Immunoblot analysis using FLAG antibody (anti-FLAG)
indicates that equivalent amounts of FLAG-WT-hSMG-1 and FLAG-KD-hSMG-1
were analyzed. D, the data presented in C are
plotted as the percent PHAS-1 phosphorylation versus
wortmannin concentration. The IC50 of hSMG-1 for wortmannin
is 105 nM using PHAS-1 as substrate. E,
top panel, HEK-293 cells were transfected with pCI-neo (
),
pCI-FLAG-WT-hSMG-1 (WT), or pCI-FLAG-KD-hSMG-1
(KD). FLAG-tagged proteins were immunopurified and incubated
with rapamycin alone (R), rapamycin-FKBP12 complex
(R:F), FK506 alone (FK), or FK506-FKBP12 complex
(FK:F) for 1 h prior to assay for autophosphorylation
and phosphorylation of PHAS-1. Immunoblot analysis using FLAG antibody
(anti-FLAG) demonstrates the presence of FLAG-hSMG-1 in the
reactions. Bottom panel, mTOR was immunopurified from
HEK-293 cells using mTAb1 antibody and incubated with either rapamycin
alone (R) or rapamycin-FKBP12 complex (R:F) for
1 h prior to assay for protein kinase activity using PHAS-1 as
substrate ([32P]PHAS-1). Immunoblot
analysis using mTAb1 antibody demonstrates the presence of mTOR in the
reactions (anti-mTAb1).
View larger version (31K):
[in a new window]
Fig. 4.
FLAG-hSMG-1 phosphorylates hUpf1 on sites
phosphorylated in whole cells. A, HEK-293 cells were
transfected with pCI-neo ( ), pCI-FLAG-WT-hSMG-1 (WT), or
pCI-FLAG-KD-hSMG-1 (KD), and FLAG-hSMG-1 protein kinase
activity toward immunopurified FLAG-hUpf1
([32P]hUpf1p) was assessed. Immunoblot
analysis using FLAG M2 monoclonal antibody demonstrates the amount
FLAG-hSMG-1 (anti-FLAG(hSMG-1)) and FLAG-hUpf1
(anti-FLAG(hUpf1p)) present in each reaction. B,
HEK-293 cells were transfected with pCI-FLAG-hUpf1, labeled for 24 h with [32P]orthophosphate and 32P-labeled
FLAG-hUpf1 immunopurified. 32P-Labeled FLAG-hUpf1
phosphorylated by FLAG-hSMG-1 in vitro (in vitro)
and 32P-labeled FLAG-hUpf1 from radiolabeled HEK-293 cells
(whole cells) were subjected to comparative two-dimensional
tryptic phosphopeptide mapping. 32P-Labeled phosphopeptides
from whole cell labeling and in vitro phosphorylation were
mixed prior to analysis to confirm the identity of phosphopeptides 1 and 2 (mix).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Jianlin Wang for technical assistance on the Northern blot analyses, Dr. Thomas Wood for advice on cloning, Esther Surriga and Steve Smith for sequencing expertise, and Dr. John C. Lawrence (University of Virginia Medical School) for supplying the rapamycin, FKBP12, FK506, antibody to mTOR, and for helpful advice.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R01 CA56869 (to A. P. F.) and DK33938 and GM59614 (to L. E. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AY014957.
** To whom correspondence should be addressed: The Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX, 77555-1048. Tel.: 409-772-1935; Fax: 409-772-1938; E-mail: afields@utmb.edu.
Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.C100144200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PIK, phosphatidylinositol kinase; PI, phosphatidylinositol; PI3K, phosphatidylinositol 3-kinase; SMG, suppressor with morphogenetic effect on genitalia; hSMG-1, human SMG-1; Upf, up frameshift; NMD, nonsense-mediated mRNA decay; TOR, targets of rapamycin; DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase; RACE, rapid amplification of cDNA ends; kb, kilobase pair(s); PCR, polymerase chain reaction; EST, expressed sequence tag; KD, kinase-deficient; GST, glutathione S-transferase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; dH2O, distilled H2O; LIP, lambda-interacting protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Wymann, M. P., and Pirola, L. (1998) Biochim. Biophys. Acta 1436, 127-150[Medline] [Order article via Infotrieve] |
2. | Keith, C. T., and Schreiber, S. L. (1995) Science 270, 50-51[Medline] [Order article via Infotrieve] |
3. | Hunter, T. (1995) Cell 83, 1-4[Medline] [Order article via Infotrieve] |
4. |
Cliby, W. A.,
Roberts, C. J.,
Cimprich, K. A.,
Stringer, C. M.,
Lamb, J. R.,
Schreiber, S. L.,
and Friend, S. H.
(1998)
EMBO J.
17,
159-169 |
5. | Bentley, N. J., and Carr, A. M. (1997) Biol. Chem. 378, 1267-1274[Medline] [Order article via Infotrieve] |
6. | Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S. H. (1994) Cell 78, 35-43[Medline] [Order article via Infotrieve] |
7. | Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N. R., and Hall, M. N. (1993) Cell 73, 585-596[Medline] [Order article via Infotrieve] |
8. |
Vilella-Bach, M.,
Nuzzi, P.,
Fang, Y.,
and Chen, J.
(1999)
J. Biol. Chem.
274,
4266-4272 |
9. | Hartley, K. O., Gell, D., Smith, G. C., Zhang, H., Divecha, N., Connelly, M. A., Admon, A., Lees-Miller, S. P., Anderson, C. W., and Jackson, S. P. (1995) Cell 82, 849-856[Medline] [Order article via Infotrieve] |
10. |
Page, M. F.,
Carr, B.,
Anders, K. R.,
Grimson, A.,
and Anderson, P.
(1999)
Mol. Cell. Biol.
19,
5943-5951 |
11. | Maquat, L. E., and Carmichael, G. G. (2001) Cell 104, 173-176[CrossRef][Medline] [Order article via Infotrieve] |
12. | Maquat, L. E. (1995) RNA (N. Y.) 1, 453-465[Abstract] |
13. | Culbertson, M. R. (1999) Trends Genet. 15, 74-80[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Frischmeyer, P. A.,
and Dietz, H. C.
(1999)
Hum. Mol. Genet.
8,
1893-1900 |
15. | Hentze, M. W., and Kulozik, A. E. (1999) Cell 96, 307-310[Medline] [Order article via Infotrieve] |
16. | Jacobson, A., and Peltz, S. W. (1996) Annu. Rev. Biochem. 65, 693-739[CrossRef][Medline] [Order article via Infotrieve] |
17. | Li, S., and Wilkinson, M. F. (1998) Immunity 8, 135-141[Medline] [Order article via Infotrieve] |
18. | Hilleren, P., and Parker, R. (1999) Annu. Rev. Genet. 33, 229-260[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Lelivelt, M. J.,
and Culbertson, M. R.
(1999)
Mol. Cell. Biol.
19,
6710-6719 |
20. |
Lew, J. E.,
Enomoto, S.,
and Berman, J.
(1998)
Mol. Cell. Biol.
18,
6121-6130 |
21. |
Mitrovich, Q. M.,
and Anderson, P.
(2000)
Genes Dev.
14,
2173-2184 |
22. |
Moriarty, P. M.,
Reddy, C. C.,
and Maquat, L. E.
(1998)
Mol. Cell. Biol.
18,
2932-2939 |
23. | Pulak, R., and Anderson, P. (1993) Genes Dev. 7, 1885-1897[Abstract] |
24. |
Pal, M.,
Ishigaki, Y.,
Nagy, E.,
and Maquat, L. E.
(2001)
RNA (N. Y.)
7,
5-15 |
25. |
Murray, N. R.,
Baumgardner, G. P.,
Burns, D. J.,
and Fields, A. P.
(1993)
J. Biol. Chem.
268,
15847-15853 |
26. |
Sun, X.,
Perlick, H. A.,
Dietz, H. C.,
and Maquat, L. E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10009-10014 |
27. |
Brunn, G. J.,
Fadden, P.,
Haystead, T. A.,
and Lawrence, J. C.
(1997)
J. Biol. Chem.
272,
32547-32550 |
28. |
Hocevar, B. A.,
Burns, D. J.,
and Fields, A. P.
(1993)
J. Biol. Chem.
268,
7545-7552 |
29. | Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Methods Enzymol. 201, 110-149[Medline] [Order article via Infotrieve] |
30. | Ishikawa, K., Nagase, T., Nakajima, D., Seki, N., Ohira, M., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., and Ohara, O. (1997) DNA Res. 4, 307-313[Medline] [Order article via Infotrieve] |
31. | Bosotti, R., Isacchi, A., and Sonnhammer, E. L. (2000) Trends Biochem. Sci. 7, 225-227[CrossRef] |
32. | Chen, J., Zheng, X. F., Brown, E. J., and Schreiber, S. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4947-4951[Abstract] |
33. | Diaz-Meco, M. T., Municio, M. M., Sanchez, P., Lozano, J., and Moscat, J. (1996) Mol. Cell. Biol. 16, 105-114[Abstract] |
34. | Sarkaria, J. N., Tibbetts, R. S., Busby, E. C., Kennedy, A. P., Hill, D. E., and Abraham, R. T. (1998) Cancer Res. 58, 4375-4382[Abstract] |
35. |
Wright, J. A.,
Keegan, K. S.,
Herendeen, D. R.,
Bentley, N. J.,
Carr, A. M.,
Hoekstra, M. F.,
and Concannon, P.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7445-7450 |
36. | Bentley, N. J., Holtzman, D. A., Flaggs, G., Keegan, K. S., DeMaggio, A., Ford, J. C., Hoekstra, M., and Carr, A. M. (1996) EMBO J. 15, 6641-6651[Abstract] |
37. | Brunn, G. J., Williams, J., Sabers, C., Wiederrecht, G., Lawrence, J. C. J., and Abraham, R. T. (1996) EMBO J. 15, 5256-5267[Abstract] |
38. | Wymann, M. P., Bulgarelli-Leva, G., Zvelebil, M. J., Pirola, L., Vanhaesebroeck, B., Waterfield, M. D., and Panayotou, G. (1996) Mol. Cell. Biol. 16, 1722-1733[Abstract] |
39. | Cutler, N. S., Heitman, J., and Cardenas, M. E. (1999) Mol. Cell. Endocrinol. 155, 135-142[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Brunn, G. J.,
Hudson, C. C.,
Sekulic, A.,
Williams, J. M.,
Hosoi, H.,
Houghton, P. J.,
Lawrence, J. C.,
and Abraham, R. T.
(1997)
Science
277,
99-101 |
41. |
Perlick, H. A.,
Medghalchi, S. M.,
Spencer, F. A.,
Kendzior, R. J.,
and Dietz, H. C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10928-10932 |
42. |
Applequist, S. E.,
Selg, M.,
Raman, C.,
and Jack, H. M.
(1997)
Nucleic Acids Res.
25,
814-821 |