Cloning of a Novel Phosphatidylinositol Kinase-related Kinase

CHARACTERIZATION OF THE HUMAN SMG-1 RNA SURVEILLANCE PROTEIN*

Gabriela DenningDagger §, Lee Jamieson§, Lynne E. Maquat, E. Aubrey ThompsonDagger §, and Alan P. FieldsDagger §||**

From the Dagger  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 (lambda -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).

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. beta -Actin mRNA served as a control for RNA loading using human beta -actin cDNA as probe (CLONTECH Laboratories Inc.). Probes were 32P-labeled by random priming using [alpha -32P]dCTP and the Megaprimer DNA labeling kit (Amersham Pharmacia Biotech).

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-beta -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.

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 -20 °C in PBS containing 10% glycerol.

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 beta -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 beta -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.

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 [gamma -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.

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.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.


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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.

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 PKCiota /lambda (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).

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.


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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 beta -actin probe is shown as a control for RNA loading (beta -actin).

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).


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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).

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.


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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).

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.

    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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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