Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI
2000; Springer Nature; Volume: 19; Issue: 3 Linguagem: Inglês
10.1093/emboj/19.3.434
ISSN1460-2075
AutoresBrian Raught, Anne‐Claude Gingras, Steven P. Gygi, Hiroaki Imataka, Shigenobu Morino, Alessandra Gradi, Ruedi Aebersold, Nahum Sonenberg,
Tópico(s)RNA modifications and cancer
ResumoArticle1 February 2000free access Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI Brian Raught Brian Raught Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Anne-Claude Gingras Anne-Claude Gingras Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Steven P. Gygi Steven P. Gygi Department of Molecular Biotechnology, University of Washington, Seattle, WA, 98195-7730 USA Search for more papers by this author Hiroaki Imataka Hiroaki Imataka Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Shigenobu Morino Shigenobu Morino Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Alessandra Gradi Alessandra Gradi Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Ruedi Aebersold Ruedi Aebersold Department of Molecular Biotechnology, University of Washington, Seattle, WA, 98195-7730 USA Search for more papers by this author Nahum Sonenberg Corresponding Author Nahum Sonenberg Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Brian Raught Brian Raught Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Anne-Claude Gingras Anne-Claude Gingras Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Steven P. Gygi Steven P. Gygi Department of Molecular Biotechnology, University of Washington, Seattle, WA, 98195-7730 USA Search for more papers by this author Hiroaki Imataka Hiroaki Imataka Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Shigenobu Morino Shigenobu Morino Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Alessandra Gradi Alessandra Gradi Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Ruedi Aebersold Ruedi Aebersold Department of Molecular Biotechnology, University of Washington, Seattle, WA, 98195-7730 USA Search for more papers by this author Nahum Sonenberg Corresponding Author Nahum Sonenberg Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Author Information Brian Raught1, Anne-Claude Gingras1, Steven P. Gygi2, Hiroaki Imataka1, Shigenobu Morino1, Alessandra Gradi1, Ruedi Aebersold2 and Nahum Sonenberg 1 1Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec, H3G 1Y6 Canada 2Department of Molecular Biotechnology, University of Washington, Seattle, WA, 98195-7730 USA ‡B.Raught and A.-C.Gingras contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:434-444https://doi.org/10.1093/emboj/19.3.434 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two-dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C-terminal one-third of the protein. Mass spectrometry and mutational analyses identified the serum-stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide-3-kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12–rapamycin-associated protein/mammalian target of rapamycin), prevent the serum-induced phosphorylation of these residues. Finally, the phosphorylation state of N-terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C-terminus to kinases is modulated by this pathway(s). Introduction A key regulatory step in protein synthesis is the recruitment of the translational machinery to the 5′ end of mRNA. This bridging process is accomplished via multiple protein–protein and protein–RNA interactions, coordinated primarily by two large, modular scaffolding factors: the eukaryotic translation initiation factor 4G (eIF4G) proteins (for reviews, see Hentze, 1997; Morley et al., 1997; Gingras et al., 1999b). eIF4GI was originally identified as a constituent of eIF4F, a tripartite complex also containing an mRNA 5′ cap binding protein, eIF4E, and an RNA helicase, eIF4A (reviewed in Gingras et al., 1999b). Viral proteases cleave the mammalian eIF4GI protein into three fragments (N-terminal, middle and C-terminal; Lamphear et al., 1995); biochemical and functional analyses of these fragments have clarified the role that each plays in the formation of a functional translation initiation complex. The N-terminal fragment interacts directly with eIF4E (Lamphear et al., 1995; Mader et al., 1995) and the poly(A) binding protein (PABP; Imataka et al., 1998). The middle fragment of eIF4GI possesses binding sites for eIF3, a multisubunit factor associated with the 40S ribosomal subunit, and eIF4A (Lamphear et al., 1995; Imataka and Sonenberg, 1997). The middle fragment also possesses RNA binding activity (Pestova et al., 1996). The C-terminal fragment of eIF4GI contains a second, independent binding site for eIF4A (Lamphear et al., 1995; Imataka and Sonenberg, 1997), and interacts with Mnk1 (MAP kinase signal-integrating kinase 1; Fukunaga and Hunter, 1997; or MAP kinase-interacting kinase 1; Waskiewicz et al., 1997), a MAP kinase-activated serine/threonine kinase that phosphorylates eIF4E in vitro (Waskiewicz et al., 1997) and in vivo (Pyronnet et al., 1999; Waskiewicz et al., 1999). A second eIF4G protein, eIF4GII, was recently characterized (Gradi et al., 1998a), which shares 46% identity with eIF4GI at the amino acid level. eIF4GI and eIF4GII are functional homologs, in that all of the features described above for eIF4GI are conserved in eIF4GII (Gradi et al., 1998a; Imataka et al., 1998; Pyronnet et al., 1999). Through the aforementioned protein–protein and protein–RNA interactions, the eIF4G proteins perform several critical functions in translation initiation, including: (i) recruitment of the 40S ribosomal subunit to the 5′ end of mRNA via interactions with eIF4E and eIF3; (ii) relief of inhibitory secondary structure in the mRNA 5′ UTR, by delivering the eIF4A helicase to this region (Rozen et al., 1990); (iii) circularization of mRNA through interactions with both eIF4E and PABP (Wells et al., 1998); and (iv) delivery of the kinase Mnk1 to its substrate, eIF4E (Pyronnet et al., 1999; Waskiewicz et al., 1999). How all of these functions are coordinated and regulated by the eIF4G proteins is not understood. The human eIF4G family also includes two other proteins: p97 and PAIP-1. p97 is homologous only to the C-terminal two-thirds of the eIF4Gs, and does not possess a region corresponding to the N-terminal one-third of the eIF4Gs. Like the eIF4Gs, it contains binding sites for eIF3, eIF4A and Mnk1. However, p97 does not interact with eIF4E or PABP (Imataka et al., 1997; Levy-Strumpf et al., 1997; Yamanaka et al., 1997), and inhibits both cap-dependent and cap-independent translation in vivo, presumably by forming non-functional initiation complexes (Imataka et al., 1997). A more distant eIF4G homolog, PAIP-1, possesses binding sites for PABP and eIF4A, and appears to act as a translational enhancer in vivo (Craig et al., 1998). eIF4G homologs have been identified in many species (e.g. Browning et al., 1987; Goyer et al., 1993; Morley et al., 1997; Hernandez et al., 1998). While all of the eIF4G-like proteins identified so far possess a region homologous to the middle fragment of the mammalian eIF4G proteins, the N- and C-terminal regions of these proteins diverge significantly. For example, an extended eIF4G C-terminus is a feature present only in certain organisms: the mammalian (Yan et al., 1992; Lamphear et al., 1993), Drosophila (Hernandez et al., 1998) and putative zebrafish eIF4G homologs possess an elongated C-terminal region. However, a wheat eIF4G homolog (Browning et al., 1987) possesses a much smaller C-terminal region, and the Saccharomyces cerevisiae (Goyer et al., 1993) and Schizosaccharomyces pombe (Morley et al., 1997) eIF4G homologs do not possess such a region at all. These observations suggest that the middle region of eIF4G is the ‘core’ unit required for translation. Consistent with this hypothesis, the middle fragment alone is sufficient for cap-independent translation from the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES; Pestova et al., 1996). The middle one-third of the protein plus the eIF4E binding site is the minimal fragment required for cap-dependent translation of the β-globin mRNA (Morino et al., 2000), and the middle region alone, fused to iron regulatory protein-1, can direct translation of an RNA containing an iron response element (DeGregorio et al., 1999). Thus, the termini of the mammalian eIF4GI protein are largely dispensable for translation. However, recent evidence suggests that both the N- and C-termini play important roles in enhancing and/or modulating translation in mammalian cells. For example, the eIF4G–PABP interaction, mediated by the eIF4G N-terminus (Imataka et al., 1998), is posited to inhibit the synthesis of truncated proteins from mRNAs that are not full-length (Sachs et al., 1997). The interaction between eIF4A and the eIF4GI C-terminus also appears to be important, as mutation of the C-terminal eIF4A binding site decreases the activity of eIF4GI in a reticulocyte lysate by several-fold (Morino et al., 2000). How the activity of the eIF4G termini may be regulated is not understood. The eIF4Gs are phosphoproteins (Duncan et al., 1987; Morley and Traugh, 1989, 1990; Donaldson et al., 1991; this study). However, the intracellular signaling pathways mediating eIF4G phosphorylation, and the location of the phosphorylation sites within the proteins, have remained uncharacterized. In this report, we map a set of phosphorylated residues in eIF4GI to the C-terminal one-third of the protein. Using mass spectrometry and mutational analyses, the serum-stimulated phosphorylation sites within this region were identified. An intracellular signaling pathway (or pathways) that may be dedicated to translational control (Brown and Schreiber, 1996) consists of phosphoinositide-3-kinase (PI3K), Akt [or protein kinase B (PKB)], and the FKBP12-rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR; also known as RAFT1 or RAPT1; reviewed in Lawrence and Abraham, 1997; Thomas and Hall, 1997; Abraham, 1998; Gingras et al., 1999b; Raught and Gingras, 1999). This pathway(s) regulates the activity of several translation factors. We demonstrate here that PI3K and FRAP/mTOR signaling modulates the serum-stimulated phosphorylation of the C-terminal eIF4GI sites. Interestingly, the eIF4GI N-terminus appears to modulate phosphorylation of the C-terminus, because phosphorylation of truncation mutants lacking the N-terminal region are resistant to kinase inhibitor treatment. Unexpectedly, although the related eIF4GII and p97 are also phosphoproteins, the corresponding C-terminal fragments in these proteins are not phosphorylated, suggesting that this region has undergone divergent evolution. Results Phosphopeptide mapping of eIF4GI To study the regulation of eIF4GI phosphorylation, two-dimensional phosphopeptide mapping of 32P-labeled, immunoprecipitated eIF4GI was conducted, essentially as described (Gingras et al., 1998). Briefly, human embryonic kidney 293 cells were metabolically labeled with [32P]orthophosphate. A subset of these cells was then stimulated with serum. The cells were lysed, and eIF4GI was immunoprecipitated using a polyclonal antiserum directed against the N-terminus of the protein (Craig et al., 1998). Immunoprecipitated material was subjected to SDS–8% PAGE, and electrotransferred to a nitrocellulose membrane. The membrane was autoradiographed, and the region harboring the protein of interest excised. Membrane pieces were subjected to tryptic digestion, and liberated phosphopeptides visualized by two-dimensional (thin-layer electrophoresis/thin-layer chromatography; TLE/TLC) mapping. A typical autoradiogram of 32P-labeled, immunoprecipitated, gel-purified eIF4GI is shown in Figure 1A. Cerenkov counting of excised membrane pieces harboring 32P-labeled eIF4GI revealed no significant difference in the quantity of 32P incorporated into eIF4GI isolated from serum-starved (−S) versus serum-stimulated (+S) 293 cells (n = 10). However, two-dimensional tryptic phosphopeptide mapping revealed that the relative intensity of several phosphorylated peptides was significantly altered upon serum stimulation, indicating a change in the phosphorylation status of one or more amino acid residues. A highly reproducible pattern of phosphorylated peptides was observed for eIF4GI isolated from serum-starved cells (Figure 1B). Treatment of starved cells with serum (Figure 1C), insulin (data not shown) or phorbol ester (data not shown) resulted in an increase in the phosphorylation state of several tryptic peptides (serum-stimulated phosphopeptides 1–4), along with a concomitant decrease in the phosphorylation status of several others (serum-repressed phosphopeptides 7–9). No significant change in the intensity of the remaining major phosphopeptides (Figure 1C, phosphopeptides 5 and 6) was observed. Phosphopeptide mapping of eIF4GI from logarithmically growing cells yielded the same phosphopeptide pattern observed for serum-stimulated cells (data not shown). Immunoprecipitation with antiserum directed against the C-terminus of human eIF4GI yielded identical maps, confirming that all of the observed phosphopeptides are derived from eIF4GI. Figure 1.Phosphorylation of specific sites in eIF4GI is modulated by serum. 32P-labeled eIF4GI immunoprecipitated from 293 cells starved of serum for 36 h (−Serum or −S), or starved of serum for 36 h then stimulated with serum for 30 min (+Serum or +S), was subjected to (A) SDS–8% PAGE, then (B and C) to two-dimensional tryptic peptide mapping. The directions of chromatography (vertical) and electrophoresis (horizontal) as well as the loading origin (arrow) are indicated. Major phosphopeptides are numbered. Download figure Download PowerPoint While the response of the phosphopeptides to serum stimulation was very consistent (i.e. peptides 1–4 increasing and peptides 7–9 decreasing in intensity), the degree of stimulation or repression varied from experiment to experiment. This variability appears to be due to a number of different reasons, including the confluence level and passage number of the cells, as well as the length of the starvation and stimulation periods. In general (as assessed by phosphoimaging and densitometry measurements), phosphopeptides 2 and 4 displayed the greatest sensitivity to serum stimulation (increasing in intensity ∼3- to 10-fold), while peptides 3 (increasing ∼2- to 5-fold) and 1 (increasing 1- to 3-fold; in some experiments no change was observed for peptide 1) were less responsive (data not shown). The intensity of peptide 7, in the serum-repressed group, also displayed acute sensitivity (decreasing by ∼2- to 10-fold) to the presence of serum, whereas peptides 8 and 9 (both decreasing by ∼2- to 5-fold) displayed a lower level of sensitivity. It is also noteworthy that while the eIF4GI phosphopeptide mapping pattern is quite reproducible, some variability is observed in the minor, unnumbered peptides as well as in peptides 4 and 6. This is most likely to be due to variations in the efficiency of tryptic digestion. Also, a streaked signal is observed directly above the loading origin in maps of endogenous eIF4GI (Figure 1B and C), suggesting that other peptides that are not clearly resolved by this method may be present. Nevertheless, the streaked signal and unnumbered phosphopeptides account for <10% of the total signal, as determined by phosphoimaging (data not shown). Mapping of eIF4GI phosphorylation sites To delineate the location of the eIF4GI phosphorylation sites, various influenza hemagglutinin (HA)-tagged eIF4GI protein fragments were expressed in 293T cells. (Phosphopeptide maps of eIF4GI from 293T cells are identical to those from serum-stimulated 293 cells. However, since the transfection efficiency achieved in 293T cells is much greater than that for 293 cells, they were used in all transfection protocols.) Logarithmically growing 293T cells were transiently transfected (in the presence of 10% serum), and labeled with [32P]orthophosphate. HA-tagged proteins were then immunoprecipitated with an anti-HA antibody, gel-purified and mapped. The constructs utilized in this study are shown in Figure 2F. HA-tagged eIF4GI protein fragments encompassing amino acids (aa) 614–1560 (Figure 2B) and 1045–1560 (Figure 2C) yielded phosphopeptide maps that were almost identical to the endogenous protein (Figure 2A). However, further N-terminal truncation to aa 1205 resulted in the loss of all phosphopeptides, except peptide 5 (Figure 2D). Mapping of a protein encompassing aa 1045–1372 yielded all phosphopeptides, except for peptides 5 and 6 (Figure 2E) and mapping of a fusion protein encompassing aa 1372–1560 yielded only peptide 5 (data not shown). Thus, all of the phosphopeptides mapped by this procedure were localized to the C-terminal one-third of the protein. The inability to localize peptide 6 is likely to result from differences in tryptic digestion efficiency between the larger and smaller eIF4GI fusion proteins. Interestingly, truncation of N-terminal sequences also resulted in a significant increase in the relative intensity of the serum-stimulated phosphopeptides (1–4) compared with the serum-repressed peptides (7–9; compare Figure 2A with E). Figure 2.Localization of eIF4GI phosphorylation sites. (A) Tryptic map of endogenous eIF4GI. (B–E) HA-tagged eIF4GI fusion protein fragments were expressed in 293T cells. Cells were metabolically labeled with 32P, and HA-tagged proteins immunoprecipitated, gel purified, and subjected to two-dimensional tryptic mapping. (F) Protein fragments tested in this study. The various protein binding regions of eIF4GI are indicated. Download figure Download PowerPoint The regions of eIF4GII and p97 corresponding to the eIF4GI phospho-region are not phosphorylated Alignment of the region of eIF4GI containing most of the phosphorylation sites (aa 1045–1205; hereafter referred to as the phospho-region) with the corresponding segments of two other members of the human eIF4G family, eIF4GII and p97, revealed relatively poor conservation (Figure 3A), especially compared with the surrounding regions. In fact, this region of human eIF4GI is much more closely related to the corresponding region of eIF4G proteins in other species than to the human eIF4GII and p97 proteins, sharing 93% identity with the rabbit eIF4GI protein (DDBJ/EMBL/GenBank accession No. L22090) and 47% identity with a putative zebrafish eIF4GI protein (DDBJ/EMBL/GenBank accession No. AI629389; data not shown). Thus, the newly defined phospho-region appears to have undergone divergent evolution within the human eIF4G family members. Figure 3.The phospho-region of eIF4GI is poorly conserved within the human eIF4G family. (A) An alignment through the newly defined eIF4GI phospho-region of the members of the human eIF4G family. Locations of defined protein binding sites in eIF4G proteins are indicated, as well as percentage identity to the corresponding region in eIF4GI. Below the diagram is an alignment of the amino acid sequences of eIF4GI, eIF4GII and p97 in this region. Identical residues are boxed in black, similar residues are boxed in gray. (B) Fragments encompassing the phosphorylated region of eIF4GI (aa 1035–1206) and the corresponding regions of eIF4GII (aa 1057–1225) and p97 (aa 395–547) were expressed in 293T cells (5, 10 and 20 μg of DNA transfected) as GST fusion proteins. Cells were metabolically labeled with 32P. Proteins isolated with glutathione-coupled Sepharose beads were washed, gel-purifed, and transferred to nitrocellulose. Upper panel, Western blot using anti-GST antiserum. Lower panel, direct autoradiogram of the same nitrocellulose membrane. Download figure Download PowerPoint Immunoprecipitation of eIF4GII and p97 from metabolically labeled 293 cells revealed that they are both phosphoproteins (data not shown). To determine whether eIF4GII and p97 are phosphorylated in the same region as eIF4GI, glutathione S-transferase (GST) fusion proteins encompassing fragments corresponding to the eIF4GI phospho-region (aa 1035–1206 for eIF4GI, aa 1057–1225 for eIF4GII, aa 395–547 for p97) were expressed in 293T cells, which were then metabolically labeled with [32P]orthophosphate. The transfected 293T cells were lysed, and GST fusion proteins isolated via incubation with glutathione-coupled Sepharose beads. Western blotting, using anti-GST antiserum (Morino et al., 2000), confirmed that all three GST fusion proteins were expressed (Figure 3B, upper panel), albeit at different levels. The same blot was then autoradiographed directly (Figure 3B, lower panel). Interestingly, only the GST–eIF4GI fusion protein incorporated significant amounts of 32P. (A weak background signal at approximately the same location as the GST–eIF4GI and GST–eIF4GII proteins is detected in all lanes; however, the intensity of this signal did not correlate with the amount of GST fusion protein expressed, and the signal did not co-migrate with GST–p97.) To ensure that all of the cells were efficiently metabolically labeled, endogenous eIF4GI was immunoprecipitated from the same lysates and found to incorporate similar amounts of 32P in all samples (data not shown). Thus, while both eIF4GII and p97 are phosphoproteins, the regions corresponding to the eIF4GI C-terminal phospho-region do not appear to be phosphorylated significantly in these members of the eIF4G family. Identification of the serum-inducible phosphorylation sites in eIF4GI To identify the C-terminal eIF4GI residues phosphorylated in response to serum treatment, mass spectrometric and mutational analyses were performed. Briefly, phosphopeptide mapping was conducted as above, except that eIF4GI immunoprecipitated from 2 × 108 serum-stimulated 293 cells (1/20 of which were metabolically labeled with [32P]orthophosphate) was loaded onto two TLC plates. The cellulose harboring peptides 1–4 was scraped from the plastic backing of the plates, and the phosphopeptides eluted and identified by capillary liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS). The tandem mass spectrum for phosphopeptide 1 corresponds to EAALPPVSPLK, with the phosphorylation site being Ser1192 (Figure 4B). The tandem mass spectrum for phosphopeptide 3 corresponds to SFSKEVEER, with the phosphorylation site being Ser1148 (Figure 4B). The tandem mass spectrum for phosphopeptide 4 corresponds to SSLSRER, with the phosphorylation site being Ser1108 (data not shown). Mass measurements for phosphopeptide 2 predicted the peptide SSLSR (data not shown), indicating that peptide 2 is an alternative cleavage product of peptide 4. The identity of the phosphorylated residue could not be determined for this peptide. Figure 4.Identification of serum-stimulated phosphorylation sites by mass spectrometry. (A) Tandem mass spectrum resulting from the analysis of excised phosphopeptide 1 from the map in Figure 1C. This spectrum contained sequence information for a single phosphopeptide. Two separate ion series were recorded simultaneously; b- and y-ion series represent sequencing inward from the N- and C-termini, respectively. The computer program Sequest (Eng et al., 1994) was utilized to match this tandem mass spectrum to the sequence shown, with the serine residue being phosphorylated. (B) Tandem mass spectrum resulting from the analysis of excised phosphopeptide 3. The position of the phosphorylated serine residue is indicated. Download figure Download PowerPoint To confirm the mass spectrometric results, site-directed mutagenesis of serines 1108, 1148 and 1192, along with several other phosphorylation site candidates, was performed. To simplify the cloning and mapping procedures these mutants were constructed in the background of the GST fusion protein containing only the eIF4GI phospho-region (aa 1035–1206). A typical two-dimensional phosphopeptide map of the GST–eIF4GI phospho-region protein is shown in Figure 5A. As observed for the HA-1045–1372 fragment (Figure 2E), phosphorylation of the GST fusion protein is almost exclusively limited to peptides 1–4, while serum-repressed peptides 7 and 8 are almost undetectable. A phosphopeptide that migrates in the same region as peptide 9 remains visible on most maps. A novel phosphopeptide (denoted by an asterisk, Figure 5A–D) that does not co-migrate with any endogenous eIF4GI peptides is observed in maps of these proteins, and is most likely to be derived from the GST moiety. Figure 5.Identification of serum-inducible phosphorylation sites by mutational analysis. Mutations of serine residues identified by mass spectrometry as phosphorylation sites were introduced into the eIF4GI phospho-region (aa 1035–1206)–GST fusion proteins. Proteins were expressed in 293T cells, 32P-labeled, isolated and mapped. Tryptic maps of the GST–eIF4GI (aa 1035–1206) wild-type protein (A), and point mutants Ser1108Ala (B), Ser1148Ala (C) and Ser1192Ala (D). A novel phosphopeptide that does not co-migrate with any endogenous eIF4GI peptide is indicated with an asterisk. (E) Complete mutational analysis summary. An alignment of the eIF4GI phospho-region with the corresponding regions of eIF4GII and p97. Identical residues are boxed in black, similar residues are boxed in gray. eIF4GI residues mutated to alanine are indicated by an ‘x’ or arrow. Locations of the serum-stimulated, phosphorylated residues, as well as the phosphopeptides identified in this analysis, are also indicated. Download figure Download PowerPoint Mutation of Ser1108 to alanine resulted in the loss of both peptides 2 and 4 (Figure 5B). Thus, tryptic cleavage after arginine 1109 appears to be relatively inefficient (most likely to be due to the presence of a phosphate group near the cleavage site), resulting in the formation of two tryptic peptides (SSLSR and SSLSRER) containing the same phosphoserine. Elimination of phosphopeptides 2 and 4 was specific for the Ser1108Ala mutant, as maps of Ser1105Ala and Ser1106Ala mutants (other serines present in the same tryptic peptide) retained peptides 1–4 (data not shown). Mutation of Ser1148 to alanine specifically abolished phosphopeptide 3 (Figure 5C), and mutation of Ser1192 to alanine eliminated phosphopeptide 1 (Figure 5D). Mutation to alanine of serines 1085, 1095, 1125, 1155, 1160 and 1170, or of threonines 1093, 1096 and 1140 had no effect on the phosphopeptide pattern (see Figure 5E for a summary of the mutational analysis). Thus, using a combination of mass spectrometric and mutational analyses, the serum-stimulated phosphorylation sites were identified as Ser1108, Ser1148 and Ser1192. PI3K and FRAP/mTOR signaling modulates eIF4GI phosphorylation Several translation regulatory factors have been identified as targets of the PI3K–FRAP/mTOR signaling pathway(s) (Thomas and Hall, 1997; Abraham, 1998; Gingras et al., 1999b). It was thus pertinent to test whether signaling to eIF4GI is also mediated by this pathway. Specific kinase inhibitors may be used to determine whether phosphorylation of a given protein is modulated by these kinases. Wortmannin inhibits PI3K by covalently binding to its catalytic subunit (reviewed in Ui et al., 1995), and LY294002 is a structurally unrelated, reversible PI3K inhibitor (Vlahos et al., 1994). Rapamycin is a fungal macrolide that, in a complex with the immunophilin FKBP12, binds to FRAP/mTOR to inhibit its kinase activity (Abraham, 1998). Serum-starved 293 cells metabolically labeled with [32P]orthophosphate (Figure 6A) were pre-treated (for 20 min) with kinase inhibitors, then serum was added to the culture media to a final concentration of 10%. After 30 min in the presence of serum (Figure 6B), or s
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