Heat Shock Impairs the Interaction of Cap-binding Protein Complex

THE JOURNAL OF BIOLOGICAL CHEMISTRY (C) 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 266, No 5, Issue of February 15, pp. 2789-2794,1991 Printed in U.S.A.

Heat Shock Impairs the Interaction of Cap-binding Protein Complex with 5‘ mRNA Cap*

(Received for publication, September 17,1990)

Barry J. Lamphear$$ and Richard PanniersSV From the llCancer Center and $Department of Biochemistry, University of Rochester, Rochester, New York 14642

Cell-free protein synthesizing systems prepared from heat-shocked Ehrlich cells retain the inhibition of translation that is seen at the cellular level. Re- cently, we showed that a highly purified cap-binding protein complex composed of the p220 and p28 subunits of eukaryotic initiation factor 4F, in a 1:l molar ratio, restores protein synthesis in these cell-free translation systems (Lamphear, B. J., and Panniers, R. (1990) J. Biol. Chern. 265, 5333-5336). Here we have estimated the amount of cap-binding complex in cell extracts that can restore protein synthesis in heat- shocked cells. We find reduced restoring activity in heat-shocked cell extracts. Further, less cap-binding complex can be purified by 7-methyl-guanosine tri- phosphate Sepharose affinity chromatography from heat-shocked cell extracts, and we conclude that heat shock impairs the binding of complex to 5’ mRNA cap. We have ruled out proteolysis and competitive inhibi- tors as mediators of this impairment. However we cannot distinguish between two possible explanations: (i) reduced association of p220 with p28 or (ii) a non- competitive inhibitor blocks complex binding to cap.

We have also examined the affect of heat shock on the phosphorylation state of two forms of p28, ~ 2 2 0 . ~ 2 8 complex and p28 free of p220. Both forms have reduced levels of phosphorylation during heat shock. The significance of these changes is discussed.

The heat shock response is elicited upon exposure of an organism to elevated temperatures (for reviews, see Refs. 1 and 2). The response is characterized by the enhanced synthesis of a small group of highly conserved proteins (the heat shock proteins or hsps)’ while the synthesis of most proteins is severely inhibited. The marked change in the pattern of protein synthesis results from regulatory events at several levels, including transcription, RNA processing (3, 4), and translation (5-7). This study focuses on translational control during heat shock.

In Ehrlich cells and other cells, polypeptide chain initiation is the primary site of translational control during heat shock (5, 7, 8). Modulation of the activities of at least two factors,

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 Present address: Hematology Unit, Dept. of Medicine, University of Rochester Medical Center, Rochester, NY 14642.

’ The abbreviations used are: hsp, heat shock protein; eIF, eukaryotic initiation factor; HRI, heme regulated inhibitor; HSPRS, high salt post-ribosomal supernatant; m‘GTP, 7-methyl-guanosine tri- phosphate; MOPS, 3-(N-morpholino)propanesulfonic acid; PMSF, phenylmethylsulfonylfluoride; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

eukaryotic initiation factor 2 and cap-binding complex are involved. The Met-tRNA binding factor eIF-2 becomes more phosphorylated during heat shock of Ehrlich cells and HeLa cells (9, 10). Phosphorylation of eIF-2 regulates initiation by trapping guanine nucleotide exchange factor (also referred to as eIF-2B or RF) preventing it from cycling eIF-2. GDP to eIF-2 .GTP and hence blocking the binding of Met-tRNAf (11, 12). With our current understanding of the mechanism of initiation, this modification can explain how overall rates of protein synthesis are inhibited during heat shock but cannot explain the selective translation of hsp mRNAs. Mod- ulation of a factor required for mRNA binding to ribosomes provides a possible explanation for selective translation. We and others have found that cap-binding protein complex activity is deficient in heat-shocked cells. Using in uitro assays for initiation factor activities, Duncan and Hershey (10) found heat shock inhibits eIF-(3+4F) in HeLa cells. Maroto and Sierra (13) suggested that inhibition of normal mRNA translation in heat-shocked Drosophila embryos is due to inacti- vation of cap binding factor(s) (13). Panniers et al. (14) showed that rabbit reticulocyte eIF-4F (a cap-binding complex) rescues cell-free translation systems prepared from heat- shocked Ehrlich cells. Further, protein synthesis in Ehrlich cell lysates can be restored by the addition of a cap-binding complex containing only p220 and cap-specific, p28, compo- nents of eIF-4F (15).

The phosphorylation state of the p28 subunit of the cap- binding complex may be important for translation initiation since levels of p28 phosphorylation correlate with the state of cell growth. The levels of phosphorylation of total cell cap- binding protein p28 have been shown to increase in the 3T3 cell response to mitogen stimulation (16), and the T cell response to mitogen stimulation (16), and to decrease during mitosis (17) as well as during heat shock (18).

In this report the mechanism of regulation of ~ 2 2 0 . ~ 2 8 cap-binding complex during heat shock is addressed. Data are presented that the ~ 2 2 0 . p28 complex is impaired in its ability to bind cap in heat-shocked cells. The evidence that this impairment results from dissociation of complex is discussed. In addition, heat shock-mediated changes in the phosphorylation state of p28 in the p220 .p28 complex, as well as p28 that is not associated with p220, is presented and the rela- tionship between phosphorylation of p28 and regulation during heat shock is discussed.

MATERIALS AND METHODS~

RESULTS

Protein Synthesis Restoring Actiuity in Cell Extracts-Pro- tein synthesis in heat-shocked cell lysates is severely inhibited

‘The “Materials and Methods” are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

2 789

2790 Heat Shock and Cap-binding Protein Complex

compared with control cell lysates (7, 14). To achieve control rates of protein synthesis, heat-shocked cell lysates require the addition of an mRNA cap-binding complex ( ~ 2 2 0 . ~ 2 8 ) containing the 220- and 28-kDa subunits of eIF-4F (15). To determine whether these effects result from the presence of a dominant inhibitor, cell-free protein synthesis systems from control and heat-shocked cells were mixed in varying ratios (Fig. 1). The presence of a dominant inhibitor in heat-shocked lysates will result in inhibition of control protein synthesis by small quantities of heat-shocked lysate and a concave curve. However, the opposite is observed. Small quantities of control lysate stimulate heat-shocked cell lysate protein synthesis (Fig. 1). Thus no dominant inhibitor is detectable in heat- shocked cell lysate. Instead, it appears likely that ~ 2 2 0 . ~ 2 8 complex in the control cell lysate replenishes a deficiency of complex in the heat shock cell lysate. To confirm the deficiency of cap complex, the levels of restoring activity (ability to stimulate cell-free protein synthesis in lysates prepared from heat-shocked cells) were compared in extracts prepared from control or heat-shocked cells. Post-mitochondrial supernatants were prepared from control and heat-shocked cells and their KC1 concentrations raised to 500 mM. After centrif- ugation to remove ribosomes, the supernatants (high-salt post-ribosomal supernatants (HSPRSs)) were compared for their ability to restore protein synthesis in heat-shocked cell lysates. Only as much restoring activity was found in heat- shocked cell HSPRS compared with control cell HSPRS (Table I).

Effect of Heat Shock on Purification of Cap-binding Protein Complex with m’GTP-Sepharose-The marked reduction in cap-binding protein complex activity in heat-shocked Ehrlich cells prompted us to determine how much complex can be

I I I -I -3 ” “0 25 50 75 100 CON

100 75 50 25 0 HS

Lysate Percentage PIC. 1. Effect of combining translation systoms from oon-

trol and heat-shocked Ehrlich cells. Control (Con) and heat- shocked ( H S ) cell-free systems were prepared as described under “Materials and Methods.” Heat-shocked cell lysate and control cell lysate were mixed in the proportions shown on the abscissa, and [“C] leucine incorporation in trichloroacetic acid-precipitable radioactivity was measured after incubation at 37 “C for 20 min at 80 mM KCl.

TABLE I Comparison of restoring activity of HSPRS fractions

from control and heat-shocked celk Crude extracts (HSPRS) were prepared from control and heat-

shocked Ehrlich cells as described in “Materials and Methods.” Protein Relative restoring

synthesis activity“

CPm arbitrary unitsb Control 1,066 37 “C HSPRS (3 pl) 4,317 31 37 “C HSPRS (5 pl) 7,160 57 44 “C HSPRS (3 pl) 2,047 9 44 “C HSPRS (5 pl) 3,224 21

HSPRSs of identical protein concentration were assayed as described under “Materials and Methods” for stimulation of heat- shocked Ehrlich cell-free protein synthesis (restoring activity).

One unit of restoring activity is equal to a 10% stimulation of cell-free protein synthesis as measured by [“Clleucine incorporation into trichloroacetic acid-precipitable material.

HS CON

200- - ”_ -

93- 68-

43- -- -

30-

- p220

- p28

FIG. 2. Analysis of m’GTP-binding proteins from control and heat-shocked cell HSPRSs by SDS-polyacrylamide gel electrophoresis. Equal amounts of protein from control and heat- shocked Ehrlich cell HSPRSs (1.5 X lo9 cells), prepared according to “Materials and Methods,” were loaded onto m’GTP-Sepharose, and bound material was specifically eluted with m‘GTP. Two % of cap- binding proteins obtained from both control cell (CON) and heat- shocked cell (HS) HSPRSs were analyzed by one-dimensional SDS- PAGE. Proteins were revealed by silver stain. Positions of molecular weight markers are indicated as well as the position of p220 and p28.

extracted from heat-shocked cells compared with control cells by m7GTP-Sepharose. HSPRSs were prepared and each ex- tract was passed over an m7GTP-Sepharose column. Specifi- cally bound proteins were eluted with m7GTP and electropho- resed on an SDS-polyacrylamide gel. Fig. 2 shows the relative amounts of cap-binding proteins obtained from these extracts. Indicated in the SDS-polyacrylamide gel in Fig. 2 is the position of p28 (previously identified as the cap-binding protein eIF-4E (15)), as well as the position of the p220 peptide. There is a considerable reduction in p220 (67%, as determined by laser derlsiLumeLric scans of silver-shined gel bands) ub- tained from heat-shocked cells. However, there is only a small reduction (26% in Fig. 2) in p28. This result has been repeated many times with complex (p220) reduced by 50-80% in heat- shocked cells but free p28 always reduced to a much smaller extent. The other major polypeptides purified from cell ex-

Heat Shock and Cap-binding Protein Complex 2791

tracts on m7GTP-Sepharose have previously been identified as belonging to eIF-3, a factor known to associate with the cap-binding complex (15, 19, 20). Binding of these polypeptides to m7GTP-Sepharose is reduced to about the same extent as p220 (Fig. 2), and this suggests that eIF-3 binds to cap- binding complex rather than p28 alone.

When control cells are fractionated into ribosomal salt wash and post-ribosomal supernatant fractions, virtually all p220- containing cap-binding complex is found in m7GTP-Sepha- rose-bound material from ribosomal salt wash as has been reported previously (15). To determine whether the cap complex that remains able to bind to m7GTP-Sepharose after heat shock is also found predominantly in the ribosomal salt wash, cap-binding proteins were isolated from post-ribosomal supernatant fractions and ribosomal salt wash fractions from heat-shocked and control cells. As seen in Fig. 3, all p220- containing complexes are extracted from the ribosomal salt washes prepared from both control and heat-shocked cells, and once again there is a large reduction in p220 obtained from heat-shocked cells while there is little reduction in total p28 obtained. Further, there appears to be increased association of an 80-kDa peptide with p28 in heat-shocked cell post- ribosomal supernatant. This 80-kDa band corresponds in molecular mass to eIF-4B, an initiation factor which is known to associate and copurify with eIF-4F (21, 22). The significance of the increased association of this 80-kDa protein with p28 during heat shock is not known.

The cap-binding peptide, p28, exists in at least two forms in control cells. The majority of p28 is free of p220 while a small proportion is bound to p220 in a complex ( ~ 2 2 0 . ~ 2 8 ) of 1:l molar ratio. These two forms can be separated on Mono Q as previously shown (15). Fractionation of heat-shocked

1 2

”

3 4 M

-200

cell cap-binding proteins on Mono Q shows the same two forms of p28 as seen in control cells (Fig. 4). However, there are markedly reduced levels of p220 .p28 in heat-shocked cells. Again, total p28 is reduced by only a small degree.

To ensure that the HSPRSs contained the total cellular pool of ~ 2 2 0 . ~ 2 8 , we estimated the amount of complex in washes of the nuclear pellet produced during the generation of post-mitochondrial supernatant. Nuclear washes (500 mM KCl, 0.5% Triton X-100) were chromatographed on m7GTP- Sepharose (same protocol as HRPRSs). Less than 5% of p28 or ~ 2 2 0 . ~ 2 8 is found in either control or heat-shocked cell nuclear washes (data not shown).

Effect of HRI on ~ 2 2 0 . ~ 2 8 Levels in Reticulocyte Lysate- The levels of eIF-2a phosphorylation are elevated in heat- shocked cells (9, 10). Phosphorylation of eIF-2a blocks initiation at a step prior to mRNA binding (23). Therefore, it is possible that the reduced levels of ~ 2 2 0 . p28 complex in heat- shocked cell lysates result indirectly from decreased rates of translational initiation caused by eIF-2 phosphorylation. To test this, protein synthesis in a reticulocyte lysate was inhibited with purified HRI, and the levels of p220-containing complex estimated by purification on m7GTP-Sepharose. As can be seen from Fig. 5A, enough HRI was added to inhibit protein synthesis by 95% within 20 min. Fig. 5B shows that HRI produces a reduction of ~ 2 2 0 . ~ 2 8 but only a 29% decrease compared with controls even though 43s complexes are dramatically reduced. Although we do not know why less cap complex is recovered from HRI-treated lysate, it is unlikely that eIF-Pa phosphorylation causes the larger reduction in cap-binding complexes that results from heat shock where

1 2 3 4 5 6 7 8

220-

- 93 -68

-43

p28-

FIG. 3. Analysis of m‘GTP-binding proteins from control and heat-shocked cell ribosomal salt wash and post-mitochondrial supernatant by SDS-polyacrylamide gel electrophoresis. Equal amounts of protein from control and heat-shocked Ehrlich cell post-ribosomal supernatant and ribosomal salt wash (generated from lo9 cells) prepared according to “Materials and Methods,” were loaded onto m’GTP-Sepharose, and bound material was specifically eluted

obtained from control cell (lunes 2 and 4 ) and heats-shocked cell (lanes 1 and 3 ) post-ribosomal supernatants (lunes I and 2 ) and ribosomal salt washes (lunes 3 and 4 ) were analyzed by one-dimensional SDS-PAGE. Proteins were revealed with silver stain. Molec- ular weights of protein markers (M) are indicated as well as the position of p220 and p28.

with m7CTP. Thc anmc pcrccntagcn of total cap-binding protcinn

FIG. 4. Analysis of Mono Q column peaks by SDS-polyacrylamide gel electrophoresis for comparison of ~ 2 2 0 . ~ 2 8 from control and heat-shocked Ehrlich cells. m7GTP cap-binding proteins obtained from equal amounts of protein from control and heat-shocked Ehrlich cell HSPRSs (see “Materials and Meth- ods’’) were fractionated identically by Mono Q chromatography to separate ~ 2 2 0 . ~ 2 8 from p28 not associated with p220. 20 pl of the

shocked (lunes 5-8) HSPRSs were anlayzed by one-dimensional SDS- PAGE. Proteins were revealed with silver stain. Lunes 1 and 5 represent the peak of p28 not associated with p220. Lanes 2 and 6 and lunes 3 and 7 represent the first half and the second half of p220. p28 peak, respectively. Lanes 4 and 8 represent the eIF-3 peak. Gel positions of p28 and p220 are indicated.

rcnpcctivc Munu Q pcuha uLLaiucd h u r wlltrul ( 1 u 1 ~ a 1-4) and llcut-


A. B. 1 2

Eawvw - p220 . ”

”

” _ -

3

TIME (minutes) FIG. 5. Effect of HRI inhibition of rabbit reticulocyte lysate

protein synthesis on levels of ~ 2 2 0 . ~ 2 8 complex. Cell-free protein synthesis in the reticulocyte lysate was performed as described under “Materials and Methods.” A, incorporation of [‘4C]leucine into trichloroacetic acid-precipitable radioactivity was measured for reactions run with (0) or without (A) purified active HRI. B, after 20 min, reactions were stopped on ice, and HSPRSs were prepared from the lysates as described under “Materials and Methods.” Cap-binding proteins were isolated from these HSPRSs by m’GTP-Sepharose affinity chromatography and analyzed by SDS-PAGE. 4% of total cap binding proteins obtained from both control (lune 1 ) and +HRI (lane 2 ) reactions were analyzed by one-dimensional SDS-PAGE. Proteins were revealed with silver stain. Positions of p220 and p28 are indicated.

CON HS

C. D.

I I I I

~ ~ 2 8 p28 pp28 p28 FIG. 6. Two-dimensional isoelectric focusing/SDS-poly-

acrylamide gel electrophoresis of different p28 forms from control and heat-shocked cells. Total cell cap-binding proteins ( A and B ) , free p28 (C and D), and Mono Q ~ 2 2 0 . ~ 2 8 complex ( E and F ) obtained from control and heat-shocked cells were analyzed by two-dimensional isoelectric focusing/SDS-PAGE and stained with silver as described under “Materials and Methods.” The section of the gels containing phosphorylated and unphosphorylated p28 are shown.

there is a smaller inhibition (85%) of protein synthesis. Effect of Heat Shock on Phosphorylation State of p28“cap-

binding proteins obtained by m7GTP-Sepharose chromatography from heat-shocked cell extracts, and control extracts were analyzed by two-dimensional isoelectric focusing/SDS- polyacrylamide gel electrophoresis. The area of the gels containing p28 are shown in Fig. 6. As reported by Duncan and Hershey (18) for heat-shocked HeLa cells, there is also a reduction in total p28 phosphorylation in Ehrlich cells during heat shock. In control Ehrlich cells, p28 associated with p220 is mostly phosphorylated while p28 free of p220 is only about 60% phosphorylated (15). In Fig. 6, cap-binding proteins from

control and heat-shocked cells were purified on m7GTP- Sepharose and free p28 and p220 .p28 separated by Mono Q. Phosphorylation of p28 in these two pools was analyzed by two-dimensional gel electrophoresis. From Fig. 6 it can be seen that phosphorylation of both pools of p28 is reduced during heat shock. Phosphorylation of free p28 is reduced from 66% in control cells to 38% in heat-shocked cells (Fig. 6, C and D); phosphorylation of p220-complexed p28 is reduced from 83 to 59% (Fig. 6, E and F).

DISCUSSION

Previously (15), it was postulated that association of p220 and p28 during initiation is blocked in heat-shocked cells. The data in this report are consistent with this hypothesis. Crude extracts prepared from heat-shocked cells representing total cytosolic fraction (HSPRS) have reduced restoring activity compared with control cells. This reduction in activity corresponds to reduced levels of p220 p28 complex that can be obtained from heat-shocked cells compared with control cells by m7GTP-Sepharose chromatography. This implies that heat shock inhibits translation of non-hsp mRNAs by im- pairing the binding of ~ 2 2 0 . ~ 2 8 to cap at the 5’ end of mRNAs.

This reduction in obtainable complex is not simply a result of a block in the binding of p28 to m7GTP because total p28 binding is little reduced. (Figs. 2-4). Further, when flow- through material from m7GTP-Sepharose chromatography of HSPRSs from both control and heat-shocked cells is reapplied to the cap-binding column, virtually no additional cap-binding proteins are extracted, suggesting that simple competition by some inhibitor for cap binding is not the mechanism of regulation of cap complex. I t is unlikely that proteolysis of p220 plays a role in reducing complex levels based on several lines of evidence. First, it has been shown that the heat shock inhibition of Ehrlich cell translational machinery can be reversed without the production of new proteins (7) suggesting that proteolysis of a component which is limiting for protein synthesis (18,22,24,25) has not occurred. Second, additional peptide bands corresponding to proteolytic fragments of p220 which can be found in m7GTP-Sepharose-bound material from poliovirus-infected cell extracts are not detected in m7GTP-bound material from heat-shocked cell extracts (Fig. 2, 3). Third, initial studies using Western blot analysis to measure p220 levels in extracts from cells after a 20-min exposure to either 37 or 44 “C show that there is virtually no reduction of p220 antigen in these extracts (data not shown). Therefore, proteolytic degradation of p220 cannot explain the large reduction in ~ 2 2 0 . ~ 2 8 complex during heat shock.

Two possible explanations of reduced p220. p28 cap binding are either (i) dissociation or prevention of association of complex or (ii) an inhibitor that non-competitively blocks complex binding to m7GTP. Given that p220 -p28 is thought to form during initiation, the simplest explanation is that heat shock prevents association as suggested previously (15). However, we are unable to definitively distinguish between the two mechanisms. If an inhibitor that interferes with cap binding is responsible, as in (ii), then a reduction of p28 equal to the reduction of p220 binding to m7GTP-Sepharose is expected (as the two polypeptides are complexed in a 1:l ratio (15)). But there is a large amount of free p28 relative to complexed $28, and su unly a small (3%) decrease irl p28 binding is predicted. Indeed, there is a consistent small reduction in p28 binding but this is always greater (>lo%) than expected. Hence, it appears that heat shock causes a small reduction in free p28 binding to m7GTP which prevents us from distinguishing between dissociation of complex or a

Heat Shock and Cap-binding Protein Complex 2793

direct block of complex binding to cap. Whether p220 association with m7GTP via p28 is impaired

through reduced association of p220 with p28 or through a stoichiometric inhibitor, heat shock results in reduced levels of the p220 .p28 complex that are able to bind to mRNA cap. Cap-binding protein complex is present at limiting levels in cells, and therefore this reduction is expected to inhibit initiation. Further cap-binding complex is capable of discrimi- nating between mRNAs with different degrees of 5' secondary structure which may explain the resistance of hsp mRNAs translation to inhibition during heat shock. Cap-binding protein complex binds mRNA 5' cap, and with several other initiation factors, mediates binding of the small ribosomal subunit to mRNA (22, 23). Part of the function of the factor appears to be the unwinding of mRNA secondary structure facilitating ribosome binding. As complex binding to cap is impaired during heat shock, unwinding must also be impaired. This will result in most mRNAs being translated poorly. However, mRNAs with reduced secondary structure, such as hsp mRNAs (26), will have a selective advantage. When eIF- 4F is added to heat-shocked cell lysates, protein synthesis of non-heat shock messages is stimulated to a greater degree than that of hsp mRNAs consistent with the ability of cap complex to discriminate between hsp and non-hsp mRNAs (14). Further support for cap complex-mediated selection of hsp mRNAs comes from work with poliovirus-infected cells where cap-binding protein complexes are reduced due to p220 degradation. Possibly as a result of low complex levels, there is a general correlation between resistance to the poliovirus- induced translational inhibition and reduced secondary structure (27). Also, heat-shock messages are more resistant to the inhibition of translation produced by poliovirus infection (28). An alternative hypothesis is cap independent initiation by ribosome binding internally on mRNA sequences (29). This has been supported for poliovirus message and has been suggested for messages with reduced secondary structure at their 5' ends (29-31), such as hsp mRNAs.

It has been suggested that p220 and p28 associate only transiently during binding of mRNA to 40 S ribosomal subunits (32). This implies that merely limiting translational initiation at a point prior to mRNA binding will result in reduced levels of p220 -p28 complex. This model predicts that the phosphorylation of eIF-2a will result in reduction of p220. p28 levels. Therefore, the increase in eIF-2a phosphorylation that occurs during heat shock might secondarily decrease cap- binding protein complex levels. However, protein synthesis in the reticulocyte cell-free system is severely inhibited (95%) by eIF-2a kinase (HRI) (greater inhibition than seen in 44 "C heat-shocked Ehrlich cells), while p220. p28 complex recovery is only reduced 29%. Hence, phosphorylation of eIF-2a appears unlikely to cause the larger reduction of ~ 2 2 0 . ~ 2 8 during heat shock. Although we cannot account for the small reduction in reticulocyte lysate cap complex, the data also suggests that p220 and p28 do not associate on ribosomal complexes with a p28- mRNA complex joining p220 on a 43 S ribosomal preinitiation complex (40 S + eIF-2.GTP.Met- tRNAf + eIF-3 + p220). Instead, if complex is formed transiently, it may occur at the 5' mRNA cap before binding to ribosomal subunits as suggested by Rhoads (22). Hence formation of ~ 2 2 0 . ~ 2 8 will be independent of 43 S complexes and eIF-2 phosphorylation.

The fact that total cell p28 changes phosphorylation state during several events, including heat shock, suggests the modification is important. Duncan et al. (18) reported that in heat-shocked HeLa cells, p28 obtained from post-mitochondrial supernatant is less phosphorylated. However, they did

not distinguish between the p220-associated form and the p220 free form of p28. Here we show that both forms of p28 have reduced levels of phosphorylation during heat shock. Further, we have compared preparations of ~ 2 2 0 . ~ 2 8 from control and heat-shocked cells (with different amounts of p28 phosphorylation) for their ability to stimulate heat-shocked lysate protein synthesis. The two preparations have the same specific activity (data not shown). Hence, like others we are unable to find a function for p28 phosphorylation.

Previously, we suggested phosphorylation of p28 may be important for the assembly of the p220 .p28 complex because p28 within this complex is so highly phosphorylated (15). p28 in the ~ 2 2 0 . ~ 2 8 complex from heat-shocked cells is still phosphorylated to a greater extent than free p28 obtained from heat-shocked cells. However, if p28 phosphorylation is required for association with p220, p28 in ~ 2 2 0 . ~ 2 8 from heat-shocked cells might be expected to be nearly fully phosphorylated as in control complexes. However, the reduced phosphorylation of p28 in ~ 2 2 0 . p28 from heat-shocked cells shows that presence of a p28 phosphate does not tightly correlate with p220 association. It is still possible that p28 phosphorylation is necessary for the process of formation of ~ 2 2 0 . ~ 2 8 and that the reduced phosphorylation of p28 in ~ 2 2 0 . p28 seen during heat shock is due to dephosphorylation after formation of the complex. However, it is equally likely that p28 phosphorylation is important for a later step in initiation.

Acknowledgments-We thank Dr. Rick Lloyd for polyclonal anti- serum raised against HeLa cell p220 degradation products and Dr. Edgar C. Henshaw for many helpful discussions.

REFERENCES 1. Lindquist, S. (1986) Annu. Reu. Biochem. 55, 1151-1191 2. Craig, E. A. (1985) CRC Critical Reu. Biochem. 18, 239-280 3. Yost, H. J., and Linquist, S. (1986) Science 242, 1544-1548 4. Bond, U. (1988) EMBO J. 7, 3509-3518 5. Hickey, E. D., and Weber, L. A. (1982) Biochemistry 21, 1513-

6. Bienz, M., and Gurdon, J. B. (1982) Cell 29,811-819 7. Panniers, R., and Henshaw, E. C. (1984) Eur. J. Biochem. 140,

8. de Benedetti, A., and Baglioni, C. (1982) J. Biol. Chem. 261,

9. Rowlands, A. G., Montine, K. S., Henshaw, E. C., and Panniers,

10. Duncan, R., and Hershey, J. W . B. (1984) J. Biol. Chem. 259,

11. Rowlands, A. G., Panniers, R., andHenshaw, E. C. (1988) J. Bid.

12. Ochoa, S. (1983) Arch. Biochem. Biophys. 223, 325-349 13. Maroto, F. G., and Sierra, J. M. (1988) J. Bioi. Chem. 263,

14. Panniers, R., Stewart, E. B., Merrick, W. C., and Henshaw, E. C.

15. Lamphear, B. J., and Panniers, R. (1990) J. Biol. Chem. 265,

16. Kaspar, R., Rychlik, W., White, M. W., Rhoads, R. E., and

17. Bonneau, A"., and Sonenberg, N. (1987) J. Biol. Chem. 262,

18. Duncan, R., Milburn, S. C., and Hershey, J. W. B. (1987) J. Biol. Chem. 262,380-388

19. Etchison, D., Milburn, S. C., Edery, I., Sonenberg, N., and Her- shey, J. W. B. (1982) J . Bioi. C k m . 257, 14806-14810

20. Trachsel, H., Sonenburg, N., Shatkin, A. J., Rose J. K., Leong, K., Bergmann, J. E., Gordan, J., and Baltimore, D. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 770-774

21. Grifo, J. A., Tahara, S. M., Morgan, M. A,, Shatkin, A. J., and Merrick, W . C. (1983) J. Biol. Chem. 258, 5804-5810

22. Rhoads, R. E. (1988) Trends Biochem. Sci. 13,52-56 23. Pain, V. M. (1986) Biochem. J . 235, 625-537 24. Ray, B. K., Brendler, T. G., Adya, S., Daniels-McQueen, S.,

1521

209-214

15800-15804

R. (1988) Eur. J. Biochem. 175,93-99

11882-11889

Chem. 263,5526-5533

15720-15725

(1985) J. Biol. Chem. 260,9648-9653

5333-5336

Morris, D. R. (1990) J. Biol. Chem. 265, 3619-3622

11134-11139


Miller, J. K., Hershey, J. W. B., Grifo, J. A., Merrick, W. C., and Thach, R. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,

25. Sarkar, G., Edery, I., Gallo, R., and Sonenberg, N. (1984) Biochim. Biophys. Acta 783, 122-129

26. Holmgren, R., Corses, V., Morimoto, R., Blackman, R., and Meselson, M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3775- 3778

663-667

27. Sonenberg, N. (1987) Adu. Virus Res. 33, 175 28. Munoz, A., Alonso, M. A,, and Carrasco, L. (1984) Virology 137,

150-159

29. Pelletier, J., and Sonenberg, N. (1989) J. Virol. 63, 441-444 30. Pelletier, J., and Sonenberg, N. (1988) Nature 334, 320-325 31. Herman, R. C. (1989) Trends Biochem. Sci. 14, 219-222 32. Etchison, D., and Smith, K. (1990) J. Biol. Chem. 265, 7492-

33. Panniers, L. R. V., and Clemens, M. J. (1981) J. Cell. Sci. 4 8 ,

34. Laemmli, U. K. (1970) Nature 227,680-685 35. O'Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021 36. Morrisey, J. H. (1981) Anal. Biochem. 117, 307-310 37. Scorsone, K. A., Panniers, R., Rowlands, A. G., and Henshaw, E.

C. (1987) J. Biol. Chem. 262, 14538-14543

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259-279

Supplemental material to "Heat Shock impalrs the lnleiaRion 01 cap binding protein

COmpiex with S mRNA cap'' by Barry J. Lamphear and Atchard Panniers

MATERIALS b N D YETHODS

MMerIal.

The radioactke amino acid ["C]leucine was obtained ham New England Nuclear.

Afftnzly chromatography medum m'GTP Sepharase 48 and FPLC Mono Q were obtained

from Pharmacla. Creatm~ne phosphokinase, MOPS, dithiothrwital. and potassium fluoride

were purchased hom Sigma Chemical Company. Niiraeiluicse membranes for western bianing (0.2 micron) were Obtalned from MluOnSsp Inc.

Cell Cult",.

Ehrlch ascites tumor cells were grown in spinner suspenslon cubre as descrlkd (33)

BrieRy. cells were grown at 3PC in Eagle's minimal essent~a medlum supplemented wlth

10% can serum. Cell cunures were diluted daily to 2 x 10' cellslml in ordw to maintain

exponential growth. The cells doubled in number abut every 15 hours

Mmhd 01 Tempmum C M r d

Suspension cultures (3-6 liters) were centrifuged at 500 x g lor 10 mlnutes at 4% and

cells were resuspended in minimal essentid medium supplemented wlth 8% can serum

These cultures were brought to temperature rapidly by resuspending cells to high densty

(3 X 10' cellslml) in pre-warmed media. me cells were Incubated at either 37'C or 44'C

lor 20 minutes, then cooled rapidly by pourlng over aushed ice and cold MOPS buffered

saline (20 mM MOPS, pH 7.4 (KOH). 154 mM NaCI).

Preparmlo" 01 R l m r n a l Sa Waeh and P a l R l m r n a l Supematam

10 min at 4'C and Csll pellets were washed twice With cold MOPS-buffered saline. h e final cell pellets were resuspended in an equai volume of hypotonic buffer (20 mM MOPS

(KOH), pH 7.4. 10 mM KCI, 2.5 mM magnesium acetate. 0.5 mM Om, 0.1 mM EOTA. 10

pglml apratonin. 1 pglmi pepstatin A and leupeptin. 0.2 mM PMSF. 10 mM phosphosennel

and cells were lysed with a Oounce homogenizer. The homogonates were made 50 mM

pellets were used lot the preparation d nuclear wash (desuibfd below). T h S

NaF. and 10 mM Bglycerophosphate and centrifuged at 1 O . w O x g for 10 min at 4%. The

postmilochondrm supernatant was funher centrllugsd at m,oa) x 9 for 2.0 h ~n a

Beckman Ti 70 rotor. Supmatant solution ( p m r w . 1 auwlulaml was removed and

StNed under liquid nitrogen for later use or used immedmtely lor factor purlcalion.

Wbosome pellerJ were suspended htgh san M e r (20 mM MOPS, pH 7.6, 10% (VIVI glycerol, 450500 mM KCI. 50 mM NaF. 2.5 mM magnesum acstate. 0.5 mM DTT. 0.1 mM

Chilled Ehrlich ceil suspension culture (5 x 10' cells/ml) was centrnuged a! 500 x g lor

EOTA) and centrihrged at 2CO.oa) x g for 2.0 h. The supernatant solution (rlboswnd mtI

wash) was stored under lcquid nlnogen or ussd immediately for factor purification.

Prep.mlon 01 Nuclesr Petl.1 WMh

Peilets wntamng nudei and mmchondna that were generated tom csntriiugation Of

cell homogenales were resuspended in nucleat wash bun.% ( 2 0 mM MOPS (KOHL pH 7.4,

500 mM KCI. 50 mM NaF, 0.5% Triton X-100. 2.5 mM magnesium acetale. 0 5 mM

d~lh~01hre1101. 0.1 mM EOTA. 10 eQJml aprotonin. 1 d m 1 pewtatin A and Ieupeptm. 0 2 mM

PMSF. to mM phosphosenns). by 5 SBOk89 of a Dounce hamogenirer. and centrituged at

t 0 . m x g for t o mm at 4-c. The rwning supematant (nw~ear puot wuh) was stored

under liquid nilragen and examined far levels of cap bindlng complexes later.

P m p u M b ot Hbh SaR P M R1mm.l SupIlVlUm (HSPRSI

Post mitochondrial supernatant was made 500 mM KCI, and cenlnfuged at 1O.wO x g

for t o min at 4%. The supmatant (HSPRS). was eithef used immedlately for analysis d

levels of cap binding proteins or stored under liquld nltrwen.

phosphoenolpyruvate, 0 4 mM spermldme. 46 pglml pyruvate kinase, 40 pM 19 amino

acids. and 5 pCllml (16 6 pM) ["C]leucme Samples. 10 el. were removed ham 3PC

Incubat8ons. and spotted on Whatman NO. 3MM fine< disa. Fllters were procsssed to

determine hot trichloroacetic acid precipitable radioaalvity as pmiously desaibed (15)

O m Step PurnlUtiDn 01 Ehrllch Call dF-JIeIF4F Prot.ln

One slep purilcaon wi(h m'GTP Sepharose started wflh high %ah post-ribosomal

supernatant (HSPRS) Which was diluted with three volumes 01 buffer A (20 mM MOPS, pH

7.6. 10% (viv) glycerol. 025 mM DTT 0.1 mM EOTA). Diluted HSPRS was applied to a 2

ml m'GTP Sepharose wlumn equilibrated with buffer E,,, (buffer A containing 50 mM NaF.

and 110 mM KCI) and the column was washed with buffer B,,, until optical density

returned to baselme and then washed with 5 wlumn volumes of buffer E,,, containing 100

pM GTP. Bound materlai was specltically eluted with 4 coiumn volumes of buffer B

contamng 70 pM m'GTP. Fractions containing m'GTP were pooled and analyzed by SOS

gel electrophoresis. For all comparisons of heat-shocked and Wntrol cell material. an equal amount of HSPRS protein was loaded onto m'GTP Sepharose.

Oetermlnatian of cap bindlng protein levels hom heat-shocked and Control cell nuclear

pellet washes and from reticuloqie HSPRS'r was pelformed by batch analysis. Cell

hactions were diluted with three volumes d buffer A,, combined with 1 ml m'GTF'

Sepharose (equlibrated in and allowed to rock slowly at 4% . After 1 hour. the

Sephaross was Sedlmented by a 5 minute 500 x g centrihrgation. and the unbound

material was removed me Sepharose was washed repeatedly with buffer A,,, until no

absorbance at 260 nm could be detected in the wash. The Sapharose was then washed

With 10 "01. A,,, containing 100 eM GTP and washed again as with buffer A,,, above until

no absorbance at 260 nm could be detected. Cap binding proteins were specifically eluted

with 6 volumes of buner A,,, wntalning 70 pM m'GTP. Specifically eluted proteins Were

analyzed by one and two dimensional gel electrophoresis

M O ~ O a separmlon DI cmrd oT Hellt-shOCked -11 pzzo1pz8 and hes p a

Cap binding protelns were purltied hom HSPRS's by one step puracation procedure

(above) as staning material for Mono Q wlumn chromatography. Separation of p22Olp28

and free p28 on Mono 0 was pelformed as described (15). Bnsfly. the m'GTP peak was

diluted to 50 mM KC1 with buffer 8, and loaded onto a 1 ml Mono 0 column BqU8librated

0.1% Tween 20) wlrh a slep to 250 mM KCI. T h i s was lollowsd by a 5 ml wash at 2 M mM

in buffer E,, contaimng 0.1 % Tween 20. Free p28 was eluted first (in buffer B wntalnlng

KCI. The ~ 2 2 0 1 ~ 2 6 wmplex was eluled ~n a 7 5 ml gradcent from 250 to 420 mM KCI.

Elsncaphc-ml.

SOS-PAGE (34). and two dimensional 1EFISDS.PAGE (35) were eflhel stlver (?5) or

Conmassce stained as stated. Quantltication 01 p220 and p28 was done using an LKB

scannlng laser dens?tometer Far quanimalon of sdver stained gels. n was assumed mal

phosphorylated and unphasphorylated forms 01 the same protein m the same gel stained

the Same

Rstlsulofyte CdI-F,ea Protein Symhad.

Reticulocyte @ate was prepared as described previously and protein Synthesis in the

reticulaqle lysate was lollowed as described (37). BriePy, 2 5 ml reactions COntained lm i

(40 %) of lysate. 20 mM MOPS, pH 76. 100 mM KCI. 1 5 mM magnesium 8celate. 0.25

mM On, 0.5 mM ATP. 0.1 mM GTP, 10 mM Creatine phosphate. 13 "nits CPK. 0.2 mM

spsrmidine. 100 VM 20 ammo acids, and S pCllml (16.6 eM) ["C]leucine. Samples. 10 el,

Were removed lrom 3PC incubalians, and swned on Whatman No. 3MM finer dma.

Finers were processed to determino hot lrichloroacelii actd prscipitablo radioactlvQ as

previously deswibed (14). At 20 minutes the remainder of the reactions were -led

rapidly on ice. and were ussd for preparing HSPRS'r and measuring levels d cap binding

protein complexes as described above.

Documents

Heat Shock Impairs the Interaction of Cap-binding Protein Complex