Inactivation of eIF2B and Phosphorylation of PHAS-I in Heat-shocked Rat Hepatoma Cells
1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês
10.1074/jbc.272.43.26850
ISSN1083-351X
AutoresGert C. Scheper, Jacqueline Mulder, Miranda Kleijn, Harry O. Voorma, Adri A.M. Thomas, Roel van Wijk,
Tópico(s)RNA Research and Splicing
ResumoVarious factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43 °C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures. Various factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43 °C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures. Incubating mammalian cells at elevated temperatures inhibits the translation of mRNAs (1Hickey E.D. Weber L.A. Biochemistry. 1982; 21: 1513-1521Crossref PubMed Scopus (92) Google Scholar, 2Nover L. Nover L. Heat Shock Response. CRC Press, Boca Raton, FL1991: 299-324Google Scholar, 3Rhoads R.E. Lamphear B.J. Sarnow P. Cap-independent Translation. Springer Verlag, Berlin1995: 131-153Google Scholar, 4Duncan R.F. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 271-293Google Scholar). The translational lesion in heat-shocked cells principally occurs at the initiation step of translation (1, 5–7; for review, see Ref. 8Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (636) Google Scholar). In the initiation process, the 7-methyl guanosine cap at the 5′ end of the mRNA is bound by a complex of eukaryotic initiation factors (eIFs), 1The abbreviations used are:eIF, eukaryotic initiation factor; 4E-BP, eIF4E-binding protein; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary; ISB, isoelectric focusing buffer; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; LSB, Laemmli sample buffer; PAGE, polyacrylamide gel electrophoresis; CIP, calf intestine alkaline phosphatase. 1The abbreviations used are:eIF, eukaryotic initiation factor; 4E-BP, eIF4E-binding protein; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary; ISB, isoelectric focusing buffer; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; LSB, Laemmli sample buffer; PAGE, polyacrylamide gel electrophoresis; CIP, calf intestine alkaline phosphatase. including the cap-binding protein eIF4E and the RNA helicase eIF4A. Unwinding of the RNA enables recognition of the AUG initiation codon by the 43 S ribosomal complex and delivery of initiator methionyl tRNA. Then the large ribosomal subunit joins the complex, and peptide synthesis begins (8Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (636) Google Scholar). Both the concentration and activity of protein synthesis initiation factors and the primary sequence of the 5′-untranslated region of an mRNA can affect the rates of eukaryotic translation initiation. With respect to the heat shock-induced modifications of eukaryotic protein synthesis initiation factors, it is of interest that the activity of many initiation factors can be regulated by phosphorylation. It has been shown that changes in the phosphorylation state of various of these eukaryotic initiation factors, such as eIF2, eIF2B, eIF4B, and eIF4E, could coincide with changes in protein synthesis (9Proud C.G. Curr. Top. Cell. Regul. 1992; 32: 243-369Crossref PubMed Scopus (164) Google Scholar, 10Redpath N.T. Proud C.G. Biochim. Biophys. Acta. 1994; 1220: 147-162Crossref PubMed Scopus (87) Google Scholar). The proteins eIF2 and eIF2B act to bring Met-tRNA to the 40 S ribosomal subunit: eIF2 by binding to Met-tRNA and GTP, and eIF2B by replacing GDP on eIF2 for GTP. Previous studies from several laboratories have focused on heat shock-induced phosphorylation of eIF2α (11Ernst V. Baum E.Z. Reddy P. Schlesinger M. Ashburner M. Tissieres A. Heat Shock From Bacteria to Man. Cold Spring Harbor Laboratory, Cold Spring Harbor1982: 215-225Google Scholar, 12De Benedetti A. Baglioni C. J. Biol. Chem. 1986; 261: 338-342Abstract Full Text PDF PubMed Google Scholar, 13Duncan R.F. Hershey J.W.B. J. Cell Biol. 1989; 109: 1467-1481Crossref PubMed Scopus (149) Google Scholar). However, it was found that less severe heat shocks that inhibit protein synthesis by more than 707 do not elicit this phosphorylation. Duncan and Hershey (13Duncan R.F. Hershey J.W.B. J. Cell Biol. 1989; 109: 1467-1481Crossref PubMed Scopus (149) Google Scholar) suggested that eIF2B might be a key player in the inhibition of protein synthesis under mild heat shock conditions. In this paper, we have studied the effect of heat shock temperatures in the range of 41–44 °C on eIF2B activity. Phosphorylation of eIF4E appears to be required for efficient cap binding and formation of the 48 S preinitiation complex (14Hiremath L.S. Hiremath S.T. Rychlik W. Joshi S. Domier L.L. Rhoads R.E. J. Biol. Chem. 1989; 264: 1132-1138Abstract Full Text PDF PubMed Google Scholar, 15Joshi-Barve S. Rychlik W. Rhoads R.E. J. Biol. Chem. 1990; 265: 2979-2983Abstract Full Text PDF PubMed Google Scholar, 16Minich W.B. Balasta M.L. Goss D.J. Rhoads R.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7668-7672Crossref PubMed Scopus (260) Google Scholar). Previous studies have demonstrated that heat shock leads to eIF4E dephosphorylation in various cell types (13Duncan R.F. Hershey J.W.B. J. Cell Biol. 1989; 109: 1467-1481Crossref PubMed Scopus (149) Google Scholar, 17Lamphear B.J. Panniers R. J. Biol. Chem. 1990; 265: 5333-5336Abstract Full Text PDF PubMed Google Scholar, 18Lamphear B.J. Panniers R. J. Biol. Chem. 1991; 266: 2789-2794Abstract Full Text PDF PubMed Google Scholar, 19Duncan R.F. Cavener D.R. Qu S. Biochemistry. 1995; 34: 2985-2997Crossref PubMed Scopus (30) Google Scholar). However, in mammalian cells this phenomenon of dephosphorylation, similar to phosphorylation of eIF2α, mainly occurs at temperatures of 43 °C or higher while protein synthesis is inhibited already at lower temperatures (13Duncan R.F. Hershey J.W.B. J. Cell Biol. 1989; 109: 1467-1481Crossref PubMed Scopus (149) Google Scholar). Therefore, eIF4E dephosphorylation cannot be the main determinant of the inhibition of protein synthesis as induced by heat shock. However, alternative types of regulation of eIF4E activity that could be involved in the decrease of protein synthesis in heat-shocked cells have become available by recent studies, using growth factors and insulin to stimulate protein synthesis. Besides phosphorylation-dephosphorylation of eIF4E, eIF4E activity can be regulated through eIF4E-binding proteins (4E-BPs) (20Lin T.-A. Kong S. Haystead T.A.J. Pause A. Belsham G.J. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (599) Google Scholar, 21Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.-A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1053) Google Scholar). The 4E-BPs compete for a binding site on eIF4E with eIF4G, the largest subunit of the eIF4F cap-binding complex (22Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 23Mader S. Lee H. Pause A. Sonenberg N. Mol. Cell. Biol. 1995; 15: 4990-4997Crossref PubMed Google Scholar). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation of the binding proteins; enhanced phosphorylation leads to dissociation of the eIF4E·4E-BP complex, increasing the amount of eIF4E available for eIF4F formation (21Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.-A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1053) Google Scholar, 24Hu C. Pang S. Kong X. Velleca M. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3730-3734Crossref PubMed Scopus (125) Google Scholar). We have studied the effects of heat shock on one of the 4E-BPs, PHAS-I, or 4E-BP1. The strong changes in protein synthesis during mild heat shock of H35-Reuber hepatoma cells are associated with the rapid inactivation of eIF2B while an unexpected phosphorylation of PHAS-I was measured due to heat shock. This suggests that at these temperatures protein synthesis is mainly regulated by this eIF2B, and not by the activity of cap-binding complexes. Rat hepatoma Reuber H35 cells were grown in Leibowitz (L15) medium (Flow/ICN Laboratories) containing potassium penicillin G (100 units/ml), streptomycin sulfate (100 ॖg/ml), and 107 fetal calf serum (Life Technologies, Inc.). Cells were grown in 25 cm2 flasks to a confluency of 80–907. In some experiments, rat pheochromocytoma PC12, Chinese hamster lung fibroblasts CCL39, Chinese hamster ovary (CHO) cells, or mouse A14 cells, derived from NIH-3T3 cells, that overexpress the insulin receptor (25Burgering B.M.T. Medema R.H. Maassen J.A. van de Wetering M.L. van der Eb A.J. Bos J.L. EMBO J. 1991; 10: 1103-1109Crossref PubMed Scopus (208) Google Scholar) were used. PC12 cells were grown in DMEM and HAM's F12 1:1 (DF medium) with 7.57 serum. A14 and CHO cells were grown in DMEM containing 7.57 serum and CCL39 cells in DMEM with 57 serum. Heat shocks were applied by submerging the culture flasks in a heated waterbath that provides a temperature stable within 0.1 °C (S.E.). Under these conditions, temperature equilibration of the cells took about 0.5 min. Cell labeling was carried out with 7 ॖCi of [35S]methionine/cysteine (Promix, Amersham) in 2 ml of HEPES-buffered DMEM lacking methionine. After harvesting of the cells, 107 of the sample to determine [35S]methionine/cysteine incorporation by hot-TCA precipitation. eIF2·[3H]GDP complexes were made as described (26Mehta H. Woodley C.L. Wahba A.J. J. Biol. Chem. 1983; 258: 3438-3441Abstract Full Text PDF PubMed Google Scholar). Briefly, 1 pmol eIF2 was incubated with 0.2 ॖCi of [3H]GDP (∼15 pmol, 30,000 dpm/pmol) in 20 mm Tris-HCl, pH 7.6, 120 mm KCl, 17 bovine serum albumin, and 1 mm dithiothreitol. After incubation at 30 °C for 15 min, 5 mm MgCl2, 1 mm GTP, and cell extract (prepared as described below) was added. Cells were harvested in 20 mm Tris-HCl, pH 7.6, 100 mm KCl, 17 Triton X-100, 0.2 mm EDTA, 50 mm ॆ-glycerophosphate, 1 mm sodium molybdate, 107 glycerol, 4 ॖg of leupeptin/ml, 0.2 mm benzamidine, 0.2 mm sodium vanadate, and 7 mmॆ-mercaptoethanol. Cell extracts, approximately 10 ॖg total protein, were added to eIF2·[3H]GDP complexes and incubated for 15 min at 30 °C. The GDP-GTP exchange reaction was stopped by adding 1 ml of a cold wash buffer (50 mmTris-HCl, pH 7.6, 5 mm MgCl2, 100 mm KCl, and 7 mm ॆ-mercaptoethanol). The mixture was filtered through nitrocellulose filters and washed three times with the same buffer. Activity of eIF2B was determined by quantification of the amount of eIF2·[3H]GDP retained on the filter. Cells were harvested in 75 ॖl of the buffer used for determination of eIF2B activity. 2 ॖl of these samples were separated by SDS-PAGE and analyzed by Western blotting. Monoclonal antibodies against eIF2α were used to visualize the total amount of eIF2α while the amount of phosphorylated eIF2α was determined with a polyclonal antibody, which was raised against an eIF2α peptide containing a phosphorylated serine residue. This antibody was a generous gift from Dr. Gary Krause. Cells were harvested in 75 ॖl of isoelectric focusing buffer (ISB) (9.5m urea (Life Technologies, Inc.), 12 mm CHAPS (Boehringer Mannheim), 0.757 Biolytes 3/10, 2.257 Biolytes 4/6 (Bio-Rad), and 700 mm ॆ-mercaptoethanol). One-third of each sample was subjected to denaturing one-dimensional isoelectric focusing as described (27Kleijn M. Voorma H.O. Thomas A.A.M. J. Cell. Biochem. 1995; 59: 443-452Crossref PubMed Scopus (21) Google Scholar). After blotting onto polyvinylidene difluoride membranes, phosphorylated and unphosphorylated forms of eIF4E were visualized with a polyclonal antibody and an alkaline phosphatase-conjugated secondary antibody. Cells were harvested in 75 ॖl of either Laemmli sample buffer (LSB) or in ISB. The ISB samples were diluted with LSB prior to loading onto SDS-polyacrylamide gels. Approximately 157 of each sample was analyzed by SDS-PAGE (13.57). PHAS-I isoforms (20Lin T.-A. Kong S. Haystead T.A.J. Pause A. Belsham G.J. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (599) Google Scholar) were detected after blotting of the proteins to polyvinylidene difluoride membranes with a rabbit antiserum raised against bacterially expressed His-tagged PHAS-I (28Haystead T.A.J. Haystead C.M.M. Hu C. Lin T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1994; 269: 23185-23191Abstract Full Text PDF PubMed Google Scholar) and goat anti-rabbit-conjugated alkaline phosphatase. For dephosphorylation of PHAS-I with calf intestine alkaline phosphatase (CIP), cells were harvested with phosphate-buffered saline/EDTA, quickly washed with 10 mm MgCl2, 10 mm ZnCl2, 100 mm Tris-HCl, pH 8.3, and resuspended in the same buffer followed by 3 freeze-thawing cycles. The lysed cells were incubated with 1 unit of CIP at 37 °C for 1 h, and at 56 °C for 1 h after addition of another unit of CIP. Cell flasks were harvested in 50 ॖl of buffer A (20 mm Tris-HCl, pH 7.6, 17 Triton X-100, 50 mm glycerophosphate, 0.2 mm EDTA, 0.2 mm EGTA, 1 mm sodium molybdate, 4 ॖg of leupeptin/ml, 0.2 mm benzamidine, 0.2 mm sodium vanadate, 7 mm ॆ-mercaptoethanol, and 107 glycerol). Extracts were diluted 75-fold in buffer A, and 2 ॖl of this dilution (approximately 30 ng of protein) was incubated in 10-ॖl assays containing 10 mm HEPES-KOH, pH 7.5, 0.2 mm EDTA, 100 mm KAc, 10 mmMgAc2, 4 ॖg of purified rabbit reticulocyte 80 S ribosomes, 100 ॖm [γ-32P]ATP (2200 dpm/pmol specific activity) for 15 min at 30 °C. Samples were analyzed by SDS-PAGE and autoradiography of the dried gels. Quantitative data were obtained with a Phosphomager (Molecular Dynamics). This method to measure S6 kinase activity is specific for p70S6K (29Chen R.H. Blenis J. Mol. Cell. Biol. 1990; 10: 3204-3215Crossref PubMed Scopus (112) Google Scholar). Heat shock of cells leads to a rapid decrease in protein synthesis, mainly caused by inhibition of translation initiation processes (2Nover L. Nover L. Heat Shock Response. CRC Press, Boca Raton, FL1991: 299-324Google Scholar, 7Schamhart D.H.J. van Walraven H.S. Wiegant F.A.C. Linnemans W.A.M. van Rijn J. van den Berg J. van Wijk R. Radiat. Res. 1984; 98: 82-95Crossref PubMed Scopus (55) Google Scholar). The subsequent time needed for recovery of protein synthesis is correlated with the duration and severeness of the stress conditions (30Van Wijk R. van Aken H. Schamhart D.H.J. Int. J. Hyperthermia. 1993; 9: 137-150Crossref PubMed Scopus (3) Google Scholar, 31Mizzen L.A. Welch W.J. J. Cell Biol. 1988; 109: 1467-1481Google Scholar). Rat H35-Reuber hepatoma cells were used to study protein synthesis during and after heat shock and to study the effects of these heat shocks on the activity or phosphorylation of various translation initiation factors. The sensitivity of protein synthesis of H35 cells for exposure to elevated temperatures (range 41–44 °C) and the ability to recover at 37 °C from inhibition of protein synthesis are shown in Fig. 1 A. In these experiments, cells were kept at the indicated temperatures for the appropriate time and incubated with [35S]methionine/cysteine for the last 15 min. All used temperatures caused a rapid and drastic inhibition of protein synthesis of at least 807 when protein synthesis was measured during the last 15 min of the heat shock period. Even stronger inhibition was found 30 min after the stress at 43 and 44 °C while cells that were incubated at 41 or 42 °C already started to recover during this period. Cells that were exposed to mild heat shock temperatures had regained control levels of protein synthesis after 4 h while protein synthesis in cells that were shocked at 43 or 44 °C was still impaired after that time period. Because changes in the activity of GTP-GDP exchange factor eIF2B will influence translation initiation on all mRNAs, this five-subunit complex is a good candidate to be affected by heat shock. Therefore, we have determined whether changes in eIF2B activity coincide with the changes in [35S]methionine/cysteine incorporation after heat shock treatment. eIF2, bound in vitro to [3H]GDP, was added to cell lysates from cells that were heat treated similarly to the samples in Fig. 1 A. Active eIF2B in the cell extracts will exchange the labeled GDP for unlabeled GTP, which can be measured by retention of labeled complexes on nitrocellulose filters. A very good correlation exists between eIF2B activity and overall protein synthesis in heat-shocked H35 cells (Fig. 1 B). The lower heat shock temperatures of 41 and 42 °C caused a rapid and transient inhibition of eIF2B activity of approximately 707. Cells shocked at either 41 or 42 °C showed recovery of eIF2B activity immediately after the heat shock while eIF2B activity in cells exposed to 43 or 44 °C only recovered partially over a 4-h interval. Inactivation of eIF2B can occur by several mechanisms, of which phosphorylation of eIF2α and subsequent sequestering of eIF2B is the best-known. We tried to determine the extent of eIF2α phosphorylation by isoelectric focusing and Western blotting. Although these blots indicated that eIF2α phosphorylation did not occur at the lower temperatures, we could not produce a consistent result. Fortunately, we obtained an antibody from Dr. Gary Krause that specifically recognizes phosphorylated eIF2α. With this antibody, the phosphorylation state of eIF2α after heat shocks at 40.5–44 °C was determined by SDS-PAGE and Western blotting (Fig.2, bottom). To show that equal amounts of eIF2 were loaded in the separate lanes, equal amounts of the lysates were analyzed with an antibody that recognizes both forms of eIF2α (top). At 40.5 and 41 °C, no change in eIF2α phosphorylation occurred (lanes 2 and 3) while at 41 °C a major decrease in eIF2B activity was measured (Fig. 1). Further increase of the heat shock temperature resulted in a gradual increase in eIF2α phosphorylation that reached its maximal level at 42.5 °C (lanes 4–6). Phosphorylation of eIF2α was increased approximately 1.8–2.2-fold at temperatures of 41.5 °C and above. As control for the eIF2α-P antibody, purified eIF2 and purified HRI were incubated in the presence of ATP (lane 10), and its phosphorylation state was compared with untreated eIF2 (lane 9). The signal obtained with phosphorylated eIF2α-specific antibodies clearly increased after HRI treatment, showing the specificity of this antibody for the phosphorylated form of eIF2α. The results in Fig. 1 and the literature data imply that levels of protein synthesis in heat-shocked cells are governed by the activity of eIF2B. Whereas no changes were found in other initiation factors, eIF2B activity was already greatly compromised at 41 °C. Interestingly, reduced eIF2B activity, coinciding with decreased protein synthesis, was found without any changes in the eIF2α phosphorylation state. Besides a reduction of protein synthesis, the heat shock response is characterized by the onset of translation of the heat shock mRNAs. Cap-binding proteins are thought to play a role in this process, and, for example in HeLa cells, diminished phosphorylation of eIF4E was found (13Duncan R.F. Hershey J.W.B. J. Cell Biol. 1989; 109: 1467-1481Crossref PubMed Scopus (149) Google Scholar). However, this reduction was found at temperatures of 43 °C and higher. Therefore, we studied the possible involvement of eIF4E-binding proteins in inhibition of protein synthesis of H35 cells. Since cell types show different sensitivities to heat stress, we have first determined whether H35 cells showed similar eIF4E dephosphorylation characteristics as HeLa cells, which have been studied thoroughly (13Duncan R.F. Hershey J.W.B. J. Cell Biol. 1989; 109: 1467-1481Crossref PubMed Scopus (149) Google Scholar) (Fig. 3). After 30 min at 43 and 44 °C eIF4E was almost completely dephosphorylated (Fig. 3, lanes 4 and 5). No decrease in eIF4E phosphorylation was found at 41 °C (lane 2) while at 42 °C eIF4E phosphorylation was reduced by approximately 507. Nevertheless, the 41 °C did result in a severe inhibition of protein synthesis (Fig. 1). Reduced protein synthesis at mild heat shock temperatures is apparently not due to dephosphorylation of eIF4E, similar to the results obtained with HeLa cells (13Duncan R.F. Hershey J.W.B. J. Cell Biol. 1989; 109: 1467-1481Crossref PubMed Scopus (149) Google Scholar). In parallel with changes in the phosphorylation state of eIF4E, the activity of eIF4E can also be controlled by changes in the activity of the eIF4E-binding protein, PHAS-I. Binding of PHAS-I to eIF4E results in reduced availability of eIF4E for eIF4F complex formation, which is needed for cap-dependent translation (32Haghighat A. Mader S. Pause A. Sonenberg N. EMBO J. 1995; 14: 5701-5709Crossref PubMed Scopus (525) Google Scholar). The binding activity of PHAS-I is controlled by changes in the phosphorylation state of PHAS-I because the dephosphorylated forms of PHAS-I bind eIF4E more tightly. Therefore, a reduced PHAS-I phosphorylation is expected in heat-shocked cells with inhibited protein synthesis. The distribution of PHAS-I among its phosphorylated forms was determined during heat shock and recovery (Fig. 4 A). Surprisingly, heat shock led to a strong increase in the phosphorylation state of PHAS-I. PHAS-I phosphorylation was increased slightly at 42 °C while at 43 and 44 °C the highly phosphorylated forms of PHAS-I were the predominant forms as compared with the control cells (Fig. 4 A, lane 16). Phosphorylation of PHAS-I continued after the cells were returned to 37 °C (comparelanes 2 and 3, 7 and 8, and12 and 13) while during further recovery dephosphorylation occurred (lanes 4 and 5, and9 and 10). A minor shift from the ॆ-form to the more phosphorylated γ-form was found at 41 °C (not shown). The increase in phosphorylation as well as the time needed for dephosphorylation were correlated with the heat shock temperature. The inhibition of protein synthesis (Fig. 1) and the enhanced phosphorylation of PHAS-I (Fig. 4 A) in heat-shocked cells is opposite to enhanced phosphorylation of PHAS-I as found after growth factor and insulin stimulation of protein synthesis. This suggests that protein synthesis is not regulated by phosphorylation of PHAS-I under these conditions and that PHAS-I phosphorylation serves other, unknown purposes than regulation of eIF4E availability. To establish the nature of the γ′-form of PHAS-I, that was found at all three temperatures in Fig. 3 A, cell extracts were treated with the alkaline phosphatase CIP (Fig. 4 B). Thus, three forms of PHAS-I were detected in extracts from untreated cells: α, ॆ, and γ (lane 1). In heat-shocked cells, the γ′-form was found (lane 3), as in Fig. 3 A. Treatment of these extracts with CIP resulted in conversion of γ- and γ′-forms to the α- and ॆ-forms of PHAS-I (lanes 2 and4), confirming that γ′-PHAS-I was a phosphorylated form of PHAS-I. The γ′-form of PHAS-I was also detected in insulin-treated rat muscle cells (34Azpiazu I. Saltiel A.R. DePaoli-Roach A.A. Lawrence Jr J.C. J. Biol. Chem. 1996; 271: 5033-5039Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The absence of conversion of the phosphorylated ॆ-form (20Lin T.-A. Kong S. Haystead T.A.J. Pause A. Belsham G.J. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (599) Google Scholar) to the unphosphorylated α-form is not understood. Similar experiments with potato acid phosphatase also failed in dephosphorylating this ॆ-form (not shown). Four other cell lines, A14, CHO, CCL39, and PC12, were used to investigate whether the fourth form of PHAS-I was unique for rat hepatoma H35 cells or whether this highly phosphorylated form was also found in other cells after heat shock (Fig.5). In mouse fibroblastic A14 cells and rat pheochromocytoma PC12 cells, heat shock also resulted in strong phosphorylation of PHAS-I as in H35 cells. Therefore, heat shock-induced phosphorylation of PHAS-I is not restricted to H35 cells but occurs in cell lines from different origins. However, phosphorylation of PHAS-I was not found in two Chinese hamster cell lines, CHO and CCL39, upon heat shock. In these cell lines, dephosphorylation of this eIF4E binding protein occurred. These unexpected results will be discussed later. Phosphorylation of PHAS-I has been correlated with increased protein synthesis after insulin administration (34Azpiazu I. Saltiel A.R. DePaoli-Roach A.A. Lawrence Jr J.C. J. Biol. Chem. 1996; 271: 5033-5039Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Under these conditions eIF4E could not longer bind to PHAS-I, making eIF4E available for translation initiation. In contrast, heat shock increased phosphorylation of PHAS-I in H35 cells and severely inhibited protein synthesis (Figs. 1 and4 A). In Fig. 6, we determined whether heat shock induced the dissociation of eIF4E·PHAS-I complexes after heat shock of H35 cells. For comparison, CHO cells were used in which heat shock induced PHAS-I dephosphorylation. H35 and CHO cells were heat-shocked at 44 °C for 30 min and returned to 37 °C for 1 or 6 h after the heat shock. In both cell lines, a decrease of protein synthesis to 10–207 of the control cells was found after heat shock as well as in cells that were replaced at 37 °C for 1 h. After recovery for 6 h, protein synthesis in both cell lines was 70–807 compared with the untreated cells. Apparently, both cell lines have similar kinetics for the levels of protein synthesis after heat shock. Fig. 6 A, top panel, shows the pattern of PHAS-I phosphorylation in the various samples. Heat shock induced a strong increase in PHAS-I phosphorylation (as shown in Fig. 4) immediately after the heat shock (lanes 2 and 3) while a recovery period of 6 h resulted in reduced PHAS-I phosphorylation (lane 4), comparable with the control cells (lane 1). The opposite effect, although less pronounced, was found in CHO cells; heat shock resulted in dephosphorylation of PHAS-I while, after a 6-h recovery period, a slight increase in PHAS-I phosphorylation was detectable. Complexes of eIF4E and PHAS-I from these samples were purified by m7GTP-Sepharose affinity chromatography (Fig.6 A, bottom panel). Similar amounts of eIF4E were recovered in all samples showing that m7GTP-binding activity of eIF4E was not changed drastically due to heat shock (18Lamphear B.J. Panniers R. J. Biol. Chem. 1991; 266: 2789-2794Abstract Full Text PDF PubMed Google Scholar). However, in H35 cells, the ability of PHAS-I to bind eIF4E was dramatically reduced after heat shock (lanes 2 and3). Apparently, phosphorylation of PHAS-I after heat shock (lanes 1 and 2) abolished complex formation between PHAS-I and eIF4E. After the recovery period, in which PHAS-I dephosphorylation occurred, an increase in eIF4E·PHAS-I complex formation was found (lane 4). A strong increase in the binding of PHAS-I to eIF4E was found in CHO cells (lanes 6and 7). The amount of associated PHAS-I after recovery (lane 8) was somewhat higher than expected. Fig. 6 B shows a schematic representation of the quantification of the results in Fig. 6 A. Phosphorylation of PHAS-I (black bars) increased from 37 to 857 in H35 cells, with a concomitant dissociation on eIF4E·PHAS-I complexes (gray bars). In the heat-shocked cells, an approximately 10-fold decrease in PHAS-I binding was found. In the cells that were allowed to recover for 6 h, PHAS-I phosphorylation and its association with eIF4E were similar to the situation found in the non-shocked cells. The approximately 407 dephosphorylation of PHAS-I found after heat shock of CHO cells, resulted in a 7-fold increase of PHAS-I binding to eIF4E. The
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