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Phosphorylation of the p220 subunit of eIF-4F by cAMP dependent protein kinase and protein kinase C in vitro
Vol. 153, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 925-932
June 30,1988
PHOSPHORYLATION OF THE p220 SUBUNIT OF eIF-4F BY cAMP DEPENDENT PROTEIN KINASE AND PROTEIN KINASE C I N V I T R O E. Lynne McMullin 1, William E. Hogancamp2, Richard D. Abramson 3, William C. Merrick3, and Curt FL Hagedoml,2,4. Department of Medicine (Gastroenterology) and Cell Biology 1, Vanderbilt University School of Medicine, Nashville, TN 37232 Department of Medicine2, Washington University School of Medicine, St. Louis, MO 63110 Department of Biochemistry 3, Case Western Reserve University, Cleveland, OH 44106 VA Medical Center4, Nashville, TN 3 7 212 Received March 7, 1988
Changes in the extent of phosphorylation of the 25 kDa subunit of elF-4F occur during several major biological events including mitosis and heat shock in mammalian ceils and shortly after fertilization of sea urchin (Lyteehinus pictus) eggs. In vitro phosphorylation studies using highly purified protein kinases demonstrated that the 220 kDa subunit of eIF-4F was phosphorylated by cAMP dependent protein kinase, protein kinase C and probably to a lesser extent by eGMP dependent protein kinase. In addition, eIF-4A was readily phosphorylated by cAMP and eGMP dependent protein kinases whereas t>48 of eIF-4F was not. The effect of these phosphorylation events on elF-4F function, its assembly or disassembly~ susceptibility to viral initiated proteolysis or the ability of p25 to be phosphorylated at serine-53 remain to be investigate& ©1 9 8 8 A c a d e m i c Press, The=
eIF-4F is a multisubunit (p25, p48, p220) initiation factor that plays an incompletely understood role in the denaturation, or unwinding, of mRNA structure and binding of mRNA to ribosomes (1-7). The physical structure and association of eIF-4F subunits is only partially understood at this time. The 25 kDa cap binding protein subunit (p25) of elF-4F has been shown to be phosphorylated in reticulocyte lysates, HeLa cells and sea urchin eggs (8-10). Changes in the phosphorylation state of this 25 kDa protein occur following fertilization of sea urchin eggs and in mammalian cells after heat shock and during mitosis (10-12). The in situ phosphorylation site of p25 in HeLa cells and rabbit reticulocytes has recently been shown to be serine 53 (13).
* Address correspondence to: ACRE Building, Room 520, V.A. Medical Center, 24th Ave. South, Nashville, TN 37212. Abbreviations: eIF-4F, eukaryotic initiation faetor-4F; eIF-4E, eukaryotic initiation faetor-4E (or cap binding protein I); elF-4A, eukaryotic initiation factor-4A; eIF-4B, eukuryotic initiation factor4]3; p220, 220 kDa subunit of elF-4F; p48, 48 kDa subunit of elF-4F; p25, 25 kDa subunit of eIF-4F; cAK, cAMP dependent protein kinas¢; cOK, cGMP dependent protein kinas¢; PKC, protein kinasc (2; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. 0006-291X/88 $1.50 925
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Phosphorylation of the p220 and p48 subunits have not been reported. The p48 subunit of elF-4F may be the same proteinas the 46-50 kDa eIF-4A thatis capable of ATP dependent unwinding of m R N A structurebut to a lesserextentthan eIF-4F (14). The p220 subunitof eIF-4F is the target of protcolyticcleavage following poliovirus and rhinovirus infection (15-22). However, this modificationof p220 appears to accountfor only partof the translationalinhibitionof host m R N A during pollovirusinfection(19). In thisreportwe describethe in vitro phosphorylationof the p220 subunitof eIF-4F by highly purifiedc A M P dependent proteinkinase and proteinkinase C. Italso appears thatc G M P dcl~ndent proteinkinascphosphorylatesp220 but at a slowerrate.
MATERIALS AND METHODS Reagents: All reagents were from Sigma Chemical Co. (St.Louis, M O ) unless specified otherwise. Highly purifiedpreparationsof c A M P and c G M P dependent protein kinase, casein kinase II and myosin lightchain kinase (rabbitskeletalmuscle) were the generous giftsof Edwin Krebs and colleagues. Purifiedc A M P dependent proteinkinasc catalyticsubunitwas the generous giftof Jackie Corbin. Proteinkinase C preparationswere the generous giftsof Robert Bell and Wayne Anderson.
Initiation factors: elF-4F, eIF-4A and eIF-4B were purified from rabbit reticulocytes by previously described methods (3, 23-25). eIF-4E was purified fl"omhuman erythrocytes after a previously described method using m7GTP sepharose (p-aminophenyl 7-ester of m7GTP, Pharrnacia) (26). Kinase assays: Incubationswith c A M P dependent protein kinase (0.4 ~tg)were 25 gl and contained20 m M Hepes-pH 7.4, 10 m M MgCI 2, I m M DTT, 100 paM [T-32p]ATP and 3 g M c A M P (27). Incubationswere for 10 rainat 30 °C, reactionswere terminatedby the addition of Laemmli sample buffer and half of each sample was analyzed by 10% S D S - P A G E and autoradiography as describedpreviously (28). Incubationswith c G M P dependent proteinkinase (0.8 gg) were 25 ~tland contained 20 m M Hepes-pH 7.4, 90 m M MgCI2, I m M DR'r, 100 [y-32p]ATP and 5 R M c G M P (29). Incubationswere for 10 rain at 30 °C and samples were analyzed in the same manner as forthe c A M P dependent proteinkinase studies.Myosin lightchain kinase incubationswere 25 gl in volume and contained 0.5 Ltg of kinase,20 m M Hepes-pH 7.4, 10 m M MgCI 2, 1 m M DTr, 100 tiM [y-32p]A'rP, I00 g M CaCI 2 and where indicatedin the figurelegends 150 n M calmodulin (30,31). Incubationswith bo~d_nelung caseinkinase II (0.2ttg) were 25 gl in volume with 20 m M Hepes-pH 7.4, 4 m M MgCI2, 150 m M NaCl and 100 g M [T-32p]A T P (32). Incubationswere at 30 °C and samples processed as alreadydescribed. Protein kinase C incubationswere in a volume of 30 gl with 20 m M Tris-pH 7.5, 5 m M MgCI2, 0.75 m M CaCl2, 100 tiM [T-32p]ATP, and contained0.3 gg leupeptin,3 gg phosphatidylserineand 0.1 gg 1,2-diolein(33,34). Incubationswere for 10 rain.at 30 oc and were analyzed as described.
RESULTS AND DISCUSSION As a survey to determine what protein kinases might modify eIF-4F in intact tissues the following in vitro studies were done with highly purified protein kinases. Purified cAMP dependent protein kinase phosphorylated the control substmtes bovine serum albumin and histone 1 (Fig. 1, Panel A). In addition, purified rabbit reticulocyte elF-4A (46-48 kDa), elF-4B (80 kDa) 926
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Fig. 1. Phosphorylation of eIF-4A, elF-4B and p220 of elF-4F by cAMP dependent protein kinase. Panel A. Incubations were for 10 rain under the conditions described in Methods. An autoradiogram obtained after a 6 hour exposure of phosphoproteins separated by SDS-PAGE is shown. The contents of incubations were as follows: oAK alone (lane 1), bovine serum albumin (1 I.tg)plus oAK (lane 2), histone 1 (1 ~tg) plus oAK (lane 3), elF-4A (1 I.tg) plus eAK (lane 4), elF-4B (1 ktg) plus oAK (lane 5), elF-4F (1 I.tg)plus eAK (lane 6) and elF-4E (1 gg) plus oAK (lane 7). An arrow indicates phosphorylated catalytic subunit of oAK. Panel B. An autoradiogram (16 h exposure) of oAK incubated alone (lane 1) and elF-4F (2 Ixg)incubated with oAK (lane 2) are shown. An arrow indicates the location of p220 in a Coomassie Blue stained unincubated elF-4F (2 I.tg)preparation in an adjacent lane. Panel C. Phosphorylation of elF-4E was visualized by autoradiographyafter a 6 fold longer exposure than shown in panel A.
and the p220 doublet of eIF-4F were phosphorylated. The phosphorylated 220 kDa doublet corresponded to the p220 doublet of Coomassie Blue stained eIF-4F (Fig. 1, Panel B). There was no apparent phosphorylation of p25 or p48 of eIF-4F. Phosphorylation of the isolated 25 kDa subunit of eIF-4F, designated eIF-4E, was only evident with exposures of autoradiograms that were 6 fold longer (Fig. 1, Panel C).
Even with these longer exposures no significant
phosphorylation of p25 of eIF-4F was observed. Incubation of eIF-4F, eIF-4E, elF-4A or eIF-4B alone did not result in phosphorylation of these substrates (data not shown). Similar studies using highly purified cGMP dependent protein kinase indicated that eIF-4A, eIF-4B and the p220 doublet of eIF-4F were phosphorylated in vitro (Fig. 2, Panel A). The 45 kDa molecular weight phosphoprotein present in the eIF-4A plus cGMP dependent protein kinase incubations may represent a proteolytie product of eIF-4A that also serves as a substrate (Fig. 2). If this is correct, it would suggest that the proteolytic product either has additional phosphorylation sites exposed or has a lower apparent K m for cGMP dependent protein kinase than native eIF-4A. Phosphorylation of eIF-4E by cGMP dependent protein kinase was also noted. However, this apparently occurred at a slow rate relative to the phosphorylation of other substrates and in order to observe this clearly a 4 fold increase in the exposure of the autoradiogram shown was required (not shown). 927
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Fig. 2. Phospho~lation of eIF-4A and eIF-4B by cGMP dependent protein kinsse. Incubations were for 10 rain at 30 °C as described in Methods. Panel A. An autoradiogram of
phosphoproteins separated by SDS-PAGE and exposed for 16 hours is shown. Incubations contained the following: cGK alone (lane 1), histone 1 (1 ktg) plus cGK (lane 2), elF-4A (1 ~tg) plus cGK (lane 3), elF-4B (1 I.tg) plus cGK (lane 4), eIF-4F (1 p.g) plus cGK (lane 5), elF-4E (lp.g) plus eGK (lane 6). Phosphorylated elF-4A (lane 3) is identified by an arrow. Panel B. Shows a silver stain of proteins in lane 3 of panel A with eIF-4A, cGK and the 45 kDa phosphoprotein identified (p45).
Casein kinase II did phosphorylate elF-4B but did not phosphorylate p220, p48 or p25 of eIF-4F (Fig. 3, panel A). It should be noted that p25 of elF-4F could be separated from the phosphorylated 13-subunit of casein kinase II in this gel system as indicated by the relative positions of Coomassie Blue stained eIF-4F subunits in an adjacent lane. Thus it is unlikely that in our SDS-PAGE system the phosphorylated ~-subunit of casein kinase II would be mistaken for phosphorylated p25 of elF-4F. When [y-32p]GTP, instead of [y-32p]ATP, was used as a phosphate donor in casein kinase II incubations no phosphorylation of p25, p48 or p220 was apparent (Fig. 3, panel B). Other studies with highly purified myosin light chain kinase indicated that this enzyme did not phosphorylate p25, p48 or p220 of eIF-4F whereas the myosin light chain control subslrate was phosphorylated in a ealmodulin dependent manner (Fig. 3, panel (2). A Ca++ regulated protein kinasc appears to phosphorylate two 25-28 kDa mammalian stress proteins, one of which might be p25 of eIF-4F (35). In view of this obsexvation the ability of protein kinase C to phosphorylate components of eIF-4F in vitro was tested. As indicated by the autoradiogram in Figure 4 (Panel A, lanes 6 and 7) the p220 subunit of eIF-4F was phosphorylated by protein kinase C in a phospholipid dependent manner. Only with exposures that were 3 to 4 times longer than shown in Figure 4 (Panel A) was phosphorylation of p25 observed (Fig. 4, Panel B). If p25 phosphorylation has a physiologic role in vivo it seems that a mechanism to accelerate the rate of this reaction, that is absent in our purified system, would be require¢L On the other 928
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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3. Incubation of elF-4F with casein kinase II and myosin lieht chain kina~¢, Panel A. The autoradiogram shown was obtained after a 10 hour exposure of incubation contents analyzed by SDS-PAGE. The incubations contained: casein kinase II alone (lane 1), dF-4B (1.5 btg) plus kinase (lane 2), and eIF-4F (1.6 I.tg) plus kinase (lane 3). The location of p25 of eIF-4F (labeled p26 here), eIF-4B and the 13-subunit of casein kinase II as determined by protein staining are indicated. Panel B. These incubations were the same as those in Panel A except that ATP was replaced with 100 ~tM [7-32p]GTP (5 gCi). The autoradiogram was obtained following a 16 h exposure. Incubations contained casein kinase II alone (lane 1), eIF-4B plus kinase (lane 2) and elF-4F plus kinase (lane 3). Panel C. Myosin light chain kinase was incubated for I0 min with the indicated substrates under the conditions described in Methods. The autoradiogram shown was obtained after a 4 day exposure. Incubations contained: myosin light chains (I ~tg) (lane 1), kinase alone (lane 2), kinase plus calmodulin (lane 3), myosin light chains plus kinase (lane 4), myosin light chains plus kinase and caimodulin (lane 5), eIF-4F (1 p.g) plus kinase (lane 6), elF-4F plus kinase and caimodulin (lane 7), elF-4E (1 I.tg) plus kinasc (lane 8) and eIF-4E plus kinase and caimodulin (lane 9). The position of myosin light chains is indicated. Fig.
929
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A MrxlO-3 216--
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Fig. 4. Protein kinase C phosphorylates p220 of eIF-4F. Panel A. Incubations were for I0 rain at 30 °C under the conditions specified in Methods. The autoradiogram shown was obtained after a 12 hour exposure of SDS-PAGE analyzed samples. Incubations contained: histone 1 (1 I.tg) alone (lane 1), PKC alone (lane 2), histone 1 plus PKC (lane 3), historic 1 plus PKC and phospholipids (lane 4), elF-4F (1.6 I,tg) alone (lane 5), elF-4F plus PKC (lane 6), eIF-4F plus PKC and phospholipids (lane 7). Panel B. Incubations were performed as described for Panel A with phospholipids and the autoradiogram shown obtained after a 90 h exposure. Incubations contained: PKC alone (lane 1), histone 1 (1 tlg) plus PKC (lane 2), elF-4F (1.6 p.g) alone (lane 3), elF-4F plus PKC (lane 4), elF-4E (0.5 I.tg)alone (lane 5) and elF-4E plus PKC (lane 6).
hand, the phosphorylation of p220 of eIF-4F by protein kinaso C may occur at a sufficiently rapid rate for it to have a regulatory role in rive. These studies indicate that cAMP dependent protein kinase, at concentrations estimated to exist in rive, and protein ldnase C phosphorylate p220 of oIF-4F in vitro (36). The specific site(s) of phosphorylation, possible effects of these modifications on eIF-4F catalytic activity, assembly or disassembly, susceptibility to viral initiated proteolysis and their frequency in rive remain to be investigated. None of the protein kinases studied here phosphorylated p25 of eIF-4F or eIF-4E at a rapid rate in vitro. Phosphorylation of eIF-4A by both cAMP and cGMP dependent protein kinasos was noted. It has been suggested that eIF-4A is the same protein as p48 of eIF-4F, possibly differing only by a post-translational modification (14). If this is true, then our findings may be explained by either lack of access to the phospborylation site when this protein is in the eIF-4F complex or possibly the existence of only pbosphorylated t)48 in the cIF-4F complex. Either possibility might be of physiologic interest since the assembly, disassembly or catalytic activity of eIF-4F might be modulated by such events.
Phosphorylation of eIF-4A may also effect
the ability of this protein to bind to the 5' cap region of mRNAs and/or mediate the ATP dependent denaturation of mRNA smcture that this factor appears to catalyze in a less efficient 930
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manner than elF-4F (6). If elF-4F plays as critical a role in translational initiation, particularly during development as recent studies suggest, then it would not be surprising if elF-4F activity was modulated by at least several different phosphorylation events in vivo (37). ACKNOWLEDGMENTS
We thank Ors. Jackie Corbin and Thomas Soderling for their comments on the manuscript and Chris Ryan and Deborah Bielser for their technical assistance. This work was supported by the Veterans Administration, Grant IN-36-Z-#1 from the American Cancer Society, and a Washington University School of Medicine Biomedical Research Services Grant.
REFERENCES 1. Shaddn, A.J. (1985) Cell 40, 223-224. 2. Sonenberg, N., Morgan, M.A., Merrick, W.C. and Shatkin, A.J. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4843-4847. 3. Grifo, J.A., Tahara, S.M., Morgan, M.A. Shaildn, A.J. and Merrick, W.C. (1983) J. Biol. Chem. 258, 5804-5810. 4. Edery, I., Humbelin, M., Darveau, A., Lee, K.A.W., Milburn, S., Hershey, J.W.B.,Trachsel, tL and Sonenberg, N. (1983) J. Biol. Chem. 258, 11398-11403. 5. Lax, S., Fritz, W., Browning, K. and Ravel, J. (1985) Proe. Natl. Acad. Sci. U.S.A. 82, 330-333. 6. Ray, B.IC, Lawson, T.G., Kramer, LC., Cladaras, M.H., Gx'ifo, J.A., Abramson, R.D., Merdck, W.C. and Thach, R.E. (1985) J. Biol. Chem. 260, 7651-7658. 7. Sarkar, G., Edery, I., Gallo., R and Sonenberg, N. (1984) Biochem. Biophys. Acta 783, 122-129. 8. Rychlik, W., Gardner, P.R., Vanaman, T.C. and Rhoads, R.E. (1986) J. Biol. Chem. 261, 71-75. 9. Hagedom, C.H., Bielser, D., Abramson, R., Merrick, W. C. and Thach, R.E. (1985) Intematl. Cong. Bioebem. Abstract FR-171. 10. Duncan, R., Milbum, S.C., and Hershey, LW.B. (1987) J. Biol. Chem. 262, 380-388. 11. Waltz, K. and Lopo, A.C. (1987) Cold Spring Harbor Meeting on Translational Control Abstract 157. 12. Bonneau, A.M. and Sonenberg, N. (1987) J. Biol. Chem. 262, 11134-11139. 13. Rychlik, W., Russ, M.A. and Rhoads, R.E. (1987) J. Biol. Chem. 262, 10434-10437. 14. Edery, I., Humbelin, M., Darveau, A., Lee, K.A.W., Milburn, S., Hershey, J.W.B., Traehsel, H. and Sonenberg, N. (1983) J. Biol. Chem. 258, 11398-11403. 15. Hewlett, M.J., Rose, J.K. and Baltimore, D. (1976) Proe. Nail. Aead. Sci. U.S.A. 73, 327-330. 16. Traehsel, H, Sonenberg,N., Shatldn, A.J., Rose, J.K., Leong, K., Bergmann, J.E., Gordon, J. and Baltimore, D. (1980) Proc. Nail. Aead. Sei. U.S.A. 77, 770-774. 17. Tahara, S.M., Morgan, M.A. and Shatldn, A.J. (1981) J. Biol. Chem. 256, 7691-7694. 18. Etchison, D., Milburn, S.C., Edery, L, Sonenberg, N. and Hershey, J.W.B. (1982) J. Biol. Chem. 257, 14806-14810. 19. Bonneau, A.M. and Sonenberg, N. (1987) J. Virol. 61, 986-991. 20. Etchison, D and Etchison, J.R. (1987) J. Virology 61, 2702-2710. 21. Krausslieh, H.G., Nieklin, M.LH., Toyoda, H., Etehison, D. and Wimmer, E. (1987) J. Virol. 61, 2711-2718. 22. Etehison, D. and Fout, S. (1985) L Virol. 54, 634-638. 23. Merdck, W.C. (1979) Methods Enzymol. 60, 101-108. 24. Benne, R., Brown-Luedi, M.L. and Hershey, J.W.R. (1979) Methods Enzymol. 60, 15-34. 25. Traehsel, H., Emi, B., Sehreier, M.H., Braun, L. and Staehelin, T. (1979) Bioehim. Biophys. Aeta 561, 484-490. 26. Webb, N.R., Chad, R.V.J., DePillis, G., Kozarich, J.W. and Rhoads, R.E. (1984) Biochemistry 23, 177-181. 27. Carmiehael, D.F., Geahlen, R.L., Allen, S.M. and Krebs, E.G. (1982) J. Biol. Chem. 257, 10440-10445. 28. Mick, S.J., Thaeh, R.E. and I-Iagedorn, C.H. (1987) Bioehem. Biophys. Res. Comm. 150, 296-303. 931
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29. Lincoln, T. M., Dills, W.L and Corbin, J.D. (1977) J. Biol. Chem. 252, 4269-4275. 30. Takio, K., Blumenthal., D.K., Edelman, A.M., Walsh, K.A., Krebs, E.G. and Titani, IC (1985) Biochemistry 24, 6028-6037. 31. Kemp, B.E. and Pearson, R.B. (1985) J. Biol. Chem. 260, 3355-3359. 32. Kuenzel, E.A. and Krebs, E.G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 737-741. 33. Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. 34. Kraft, A.S., Anderson, W.B., Cooper, H.L. and Sando, J.J. (1982) J. Biol. Chem. 257,13193-13196. 35. Welch, W.J. (1985) J. Biol. Chem. 260, 3058-3062. 36. Corbin, J. D., Sugden, P. H., Lincoln, T.M. and Keely, S. L. (1977) J. Biol. Chem. 2S2, 3854-3861. 37. Clemens, M. (1987) Nature 330, 699-700.
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June 30,1988
PHOSPHORYLATION OF THE p220 SUBUNIT OF eIF-4F BY cAMP DEPENDENT PROTEIN KINASE AND PROTEIN KINASE C I N V I T R O E. Lynne McMullin 1, William E. Hogancamp2, Richard D. Abramson 3, William C. Merrick3, and Curt FL Hagedoml,2,4. Department of Medicine (Gastroenterology) and Cell Biology 1, Vanderbilt University School of Medicine, Nashville, TN 37232 Department of Medicine2, Washington University School of Medicine, St. Louis, MO 63110 Department of Biochemistry 3, Case Western Reserve University, Cleveland, OH 44106 VA Medical Center4, Nashville, TN 3 7 212 Received March 7, 1988
Changes in the extent of phosphorylation of the 25 kDa subunit of elF-4F occur during several major biological events including mitosis and heat shock in mammalian ceils and shortly after fertilization of sea urchin (Lyteehinus pictus) eggs. In vitro phosphorylation studies using highly purified protein kinases demonstrated that the 220 kDa subunit of eIF-4F was phosphorylated by cAMP dependent protein kinase, protein kinase C and probably to a lesser extent by eGMP dependent protein kinase. In addition, eIF-4A was readily phosphorylated by cAMP and eGMP dependent protein kinases whereas t>48 of eIF-4F was not. The effect of these phosphorylation events on elF-4F function, its assembly or disassembly~ susceptibility to viral initiated proteolysis or the ability of p25 to be phosphorylated at serine-53 remain to be investigate& ©1 9 8 8 A c a d e m i c Press, The=
eIF-4F is a multisubunit (p25, p48, p220) initiation factor that plays an incompletely understood role in the denaturation, or unwinding, of mRNA structure and binding of mRNA to ribosomes (1-7). The physical structure and association of eIF-4F subunits is only partially understood at this time. The 25 kDa cap binding protein subunit (p25) of elF-4F has been shown to be phosphorylated in reticulocyte lysates, HeLa cells and sea urchin eggs (8-10). Changes in the phosphorylation state of this 25 kDa protein occur following fertilization of sea urchin eggs and in mammalian cells after heat shock and during mitosis (10-12). The in situ phosphorylation site of p25 in HeLa cells and rabbit reticulocytes has recently been shown to be serine 53 (13).
* Address correspondence to: ACRE Building, Room 520, V.A. Medical Center, 24th Ave. South, Nashville, TN 37212. Abbreviations: eIF-4F, eukaryotic initiation faetor-4F; eIF-4E, eukaryotic initiation faetor-4E (or cap binding protein I); elF-4A, eukaryotic initiation factor-4A; eIF-4B, eukuryotic initiation factor4]3; p220, 220 kDa subunit of elF-4F; p48, 48 kDa subunit of elF-4F; p25, 25 kDa subunit of eIF-4F; cAK, cAMP dependent protein kinas¢; cOK, cGMP dependent protein kinas¢; PKC, protein kinasc (2; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. 0006-291X/88 $1.50 925
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Phosphorylation of the p220 and p48 subunits have not been reported. The p48 subunit of elF-4F may be the same proteinas the 46-50 kDa eIF-4A thatis capable of ATP dependent unwinding of m R N A structurebut to a lesserextentthan eIF-4F (14). The p220 subunitof eIF-4F is the target of protcolyticcleavage following poliovirus and rhinovirus infection (15-22). However, this modificationof p220 appears to accountfor only partof the translationalinhibitionof host m R N A during pollovirusinfection(19). In thisreportwe describethe in vitro phosphorylationof the p220 subunitof eIF-4F by highly purifiedc A M P dependent proteinkinase and proteinkinase C. Italso appears thatc G M P dcl~ndent proteinkinascphosphorylatesp220 but at a slowerrate.
MATERIALS AND METHODS Reagents: All reagents were from Sigma Chemical Co. (St.Louis, M O ) unless specified otherwise. Highly purifiedpreparationsof c A M P and c G M P dependent protein kinase, casein kinase II and myosin lightchain kinase (rabbitskeletalmuscle) were the generous giftsof Edwin Krebs and colleagues. Purifiedc A M P dependent proteinkinasc catalyticsubunitwas the generous giftof Jackie Corbin. Proteinkinase C preparationswere the generous giftsof Robert Bell and Wayne Anderson.
Initiation factors: elF-4F, eIF-4A and eIF-4B were purified from rabbit reticulocytes by previously described methods (3, 23-25). eIF-4E was purified fl"omhuman erythrocytes after a previously described method using m7GTP sepharose (p-aminophenyl 7-ester of m7GTP, Pharrnacia) (26). Kinase assays: Incubationswith c A M P dependent protein kinase (0.4 ~tg)were 25 gl and contained20 m M Hepes-pH 7.4, 10 m M MgCI 2, I m M DTT, 100 paM [T-32p]ATP and 3 g M c A M P (27). Incubationswere for 10 rainat 30 °C, reactionswere terminatedby the addition of Laemmli sample buffer and half of each sample was analyzed by 10% S D S - P A G E and autoradiography as describedpreviously (28). Incubationswith c G M P dependent proteinkinase (0.8 gg) were 25 ~tland contained 20 m M Hepes-pH 7.4, 90 m M MgCI2, I m M DR'r, 100 [y-32p]ATP and 5 R M c G M P (29). Incubationswere for 10 rain at 30 °C and samples were analyzed in the same manner as forthe c A M P dependent proteinkinase studies.Myosin lightchain kinase incubationswere 25 gl in volume and contained 0.5 Ltg of kinase,20 m M Hepes-pH 7.4, 10 m M MgCI 2, 1 m M DTr, 100 tiM [y-32p]A'rP, I00 g M CaCI 2 and where indicatedin the figurelegends 150 n M calmodulin (30,31). Incubationswith bo~d_nelung caseinkinase II (0.2ttg) were 25 gl in volume with 20 m M Hepes-pH 7.4, 4 m M MgCI2, 150 m M NaCl and 100 g M [T-32p]A T P (32). Incubationswere at 30 °C and samples processed as alreadydescribed. Protein kinase C incubationswere in a volume of 30 gl with 20 m M Tris-pH 7.5, 5 m M MgCI2, 0.75 m M CaCl2, 100 tiM [T-32p]ATP, and contained0.3 gg leupeptin,3 gg phosphatidylserineand 0.1 gg 1,2-diolein(33,34). Incubationswere for 10 rain.at 30 oc and were analyzed as described.
RESULTS AND DISCUSSION As a survey to determine what protein kinases might modify eIF-4F in intact tissues the following in vitro studies were done with highly purified protein kinases. Purified cAMP dependent protein kinase phosphorylated the control substmtes bovine serum albumin and histone 1 (Fig. 1, Panel A). In addition, purified rabbit reticulocyte elF-4A (46-48 kDa), elF-4B (80 kDa) 926
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A MrxlO'3
B M r x I0 "3
216 --
<-p220
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:+ -* elF-4E
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3
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6
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Fig. 1. Phosphorylation of eIF-4A, elF-4B and p220 of elF-4F by cAMP dependent protein kinase. Panel A. Incubations were for 10 rain under the conditions described in Methods. An autoradiogram obtained after a 6 hour exposure of phosphoproteins separated by SDS-PAGE is shown. The contents of incubations were as follows: oAK alone (lane 1), bovine serum albumin (1 I.tg)plus oAK (lane 2), histone 1 (1 ~tg) plus oAK (lane 3), elF-4A (1 I.tg) plus eAK (lane 4), elF-4B (1 ktg) plus oAK (lane 5), elF-4F (1 I.tg)plus eAK (lane 6) and elF-4E (1 gg) plus oAK (lane 7). An arrow indicates phosphorylated catalytic subunit of oAK. Panel B. An autoradiogram (16 h exposure) of oAK incubated alone (lane 1) and elF-4F (2 Ixg)incubated with oAK (lane 2) are shown. An arrow indicates the location of p220 in a Coomassie Blue stained unincubated elF-4F (2 I.tg)preparation in an adjacent lane. Panel C. Phosphorylation of elF-4E was visualized by autoradiographyafter a 6 fold longer exposure than shown in panel A.
and the p220 doublet of eIF-4F were phosphorylated. The phosphorylated 220 kDa doublet corresponded to the p220 doublet of Coomassie Blue stained eIF-4F (Fig. 1, Panel B). There was no apparent phosphorylation of p25 or p48 of eIF-4F. Phosphorylation of the isolated 25 kDa subunit of eIF-4F, designated eIF-4E, was only evident with exposures of autoradiograms that were 6 fold longer (Fig. 1, Panel C).
Even with these longer exposures no significant
phosphorylation of p25 of eIF-4F was observed. Incubation of eIF-4F, eIF-4E, elF-4A or eIF-4B alone did not result in phosphorylation of these substrates (data not shown). Similar studies using highly purified cGMP dependent protein kinase indicated that eIF-4A, eIF-4B and the p220 doublet of eIF-4F were phosphorylated in vitro (Fig. 2, Panel A). The 45 kDa molecular weight phosphoprotein present in the eIF-4A plus cGMP dependent protein kinase incubations may represent a proteolytie product of eIF-4A that also serves as a substrate (Fig. 2). If this is correct, it would suggest that the proteolytic product either has additional phosphorylation sites exposed or has a lower apparent K m for cGMP dependent protein kinase than native eIF-4A. Phosphorylation of eIF-4E by cGMP dependent protein kinase was also noted. However, this apparently occurred at a slow rate relative to the phosphorylation of other substrates and in order to observe this clearly a 4 fold increase in the exposure of the autoradiogram shown was required (not shown). 927
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A MrxlO_ 3
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B
ilBImJliE~.l~IR
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Fig. 2. Phospho~lation of eIF-4A and eIF-4B by cGMP dependent protein kinsse. Incubations were for 10 rain at 30 °C as described in Methods. Panel A. An autoradiogram of
phosphoproteins separated by SDS-PAGE and exposed for 16 hours is shown. Incubations contained the following: cGK alone (lane 1), histone 1 (1 ktg) plus cGK (lane 2), elF-4A (1 ~tg) plus cGK (lane 3), elF-4B (1 I.tg) plus cGK (lane 4), eIF-4F (1 p.g) plus cGK (lane 5), elF-4E (lp.g) plus eGK (lane 6). Phosphorylated elF-4A (lane 3) is identified by an arrow. Panel B. Shows a silver stain of proteins in lane 3 of panel A with eIF-4A, cGK and the 45 kDa phosphoprotein identified (p45).
Casein kinase II did phosphorylate elF-4B but did not phosphorylate p220, p48 or p25 of eIF-4F (Fig. 3, panel A). It should be noted that p25 of elF-4F could be separated from the phosphorylated 13-subunit of casein kinase II in this gel system as indicated by the relative positions of Coomassie Blue stained eIF-4F subunits in an adjacent lane. Thus it is unlikely that in our SDS-PAGE system the phosphorylated ~-subunit of casein kinase II would be mistaken for phosphorylated p25 of elF-4F. When [y-32p]GTP, instead of [y-32p]ATP, was used as a phosphate donor in casein kinase II incubations no phosphorylation of p25, p48 or p220 was apparent (Fig. 3, panel B). Other studies with highly purified myosin light chain kinase indicated that this enzyme did not phosphorylate p25, p48 or p220 of eIF-4F whereas the myosin light chain control subslrate was phosphorylated in a ealmodulin dependent manner (Fig. 3, panel (2). A Ca++ regulated protein kinasc appears to phosphorylate two 25-28 kDa mammalian stress proteins, one of which might be p25 of eIF-4F (35). In view of this obsexvation the ability of protein kinase C to phosphorylate components of eIF-4F in vitro was tested. As indicated by the autoradiogram in Figure 4 (Panel A, lanes 6 and 7) the p220 subunit of eIF-4F was phosphorylated by protein kinase C in a phospholipid dependent manner. Only with exposures that were 3 to 4 times longer than shown in Figure 4 (Panel A) was phosphorylation of p25 observed (Fig. 4, Panel B). If p25 phosphorylation has a physiologic role in vivo it seems that a mechanism to accelerate the rate of this reaction, that is absent in our purified system, would be require¢L On the other 928
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3. Incubation of elF-4F with casein kinase II and myosin lieht chain kina~¢, Panel A. The autoradiogram shown was obtained after a 10 hour exposure of incubation contents analyzed by SDS-PAGE. The incubations contained: casein kinase II alone (lane 1), dF-4B (1.5 btg) plus kinase (lane 2), and eIF-4F (1.6 I.tg) plus kinase (lane 3). The location of p25 of eIF-4F (labeled p26 here), eIF-4B and the 13-subunit of casein kinase II as determined by protein staining are indicated. Panel B. These incubations were the same as those in Panel A except that ATP was replaced with 100 ~tM [7-32p]GTP (5 gCi). The autoradiogram was obtained following a 16 h exposure. Incubations contained casein kinase II alone (lane 1), eIF-4B plus kinase (lane 2) and elF-4F plus kinase (lane 3). Panel C. Myosin light chain kinase was incubated for I0 min with the indicated substrates under the conditions described in Methods. The autoradiogram shown was obtained after a 4 day exposure. Incubations contained: myosin light chains (I ~tg) (lane 1), kinase alone (lane 2), kinase plus calmodulin (lane 3), myosin light chains plus kinase (lane 4), myosin light chains plus kinase and caimodulin (lane 5), eIF-4F (1 p.g) plus kinase (lane 6), elF-4F plus kinase and caimodulin (lane 7), elF-4E (1 I.tg) plus kinasc (lane 8) and eIF-4E plus kinase and caimodulin (lane 9). The position of myosin light chains is indicated. Fig.
929
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A MrxlO-3 216--
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3 4 5 6 7
.~o
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Fig. 4. Protein kinase C phosphorylates p220 of eIF-4F. Panel A. Incubations were for I0 rain at 30 °C under the conditions specified in Methods. The autoradiogram shown was obtained after a 12 hour exposure of SDS-PAGE analyzed samples. Incubations contained: histone 1 (1 I.tg) alone (lane 1), PKC alone (lane 2), histone 1 plus PKC (lane 3), historic 1 plus PKC and phospholipids (lane 4), elF-4F (1.6 I,tg) alone (lane 5), elF-4F plus PKC (lane 6), eIF-4F plus PKC and phospholipids (lane 7). Panel B. Incubations were performed as described for Panel A with phospholipids and the autoradiogram shown obtained after a 90 h exposure. Incubations contained: PKC alone (lane 1), histone 1 (1 tlg) plus PKC (lane 2), elF-4F (1.6 p.g) alone (lane 3), elF-4F plus PKC (lane 4), elF-4E (0.5 I.tg)alone (lane 5) and elF-4E plus PKC (lane 6).
hand, the phosphorylation of p220 of eIF-4F by protein kinaso C may occur at a sufficiently rapid rate for it to have a regulatory role in rive. These studies indicate that cAMP dependent protein kinase, at concentrations estimated to exist in rive, and protein ldnase C phosphorylate p220 of oIF-4F in vitro (36). The specific site(s) of phosphorylation, possible effects of these modifications on eIF-4F catalytic activity, assembly or disassembly, susceptibility to viral initiated proteolysis and their frequency in rive remain to be investigated. None of the protein kinases studied here phosphorylated p25 of eIF-4F or eIF-4E at a rapid rate in vitro. Phosphorylation of eIF-4A by both cAMP and cGMP dependent protein kinasos was noted. It has been suggested that eIF-4A is the same protein as p48 of eIF-4F, possibly differing only by a post-translational modification (14). If this is true, then our findings may be explained by either lack of access to the phospborylation site when this protein is in the eIF-4F complex or possibly the existence of only pbosphorylated t)48 in the cIF-4F complex. Either possibility might be of physiologic interest since the assembly, disassembly or catalytic activity of eIF-4F might be modulated by such events.
Phosphorylation of eIF-4A may also effect
the ability of this protein to bind to the 5' cap region of mRNAs and/or mediate the ATP dependent denaturation of mRNA smcture that this factor appears to catalyze in a less efficient 930
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manner than elF-4F (6). If elF-4F plays as critical a role in translational initiation, particularly during development as recent studies suggest, then it would not be surprising if elF-4F activity was modulated by at least several different phosphorylation events in vivo (37). ACKNOWLEDGMENTS
We thank Ors. Jackie Corbin and Thomas Soderling for their comments on the manuscript and Chris Ryan and Deborah Bielser for their technical assistance. This work was supported by the Veterans Administration, Grant IN-36-Z-#1 from the American Cancer Society, and a Washington University School of Medicine Biomedical Research Services Grant.
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29. Lincoln, T. M., Dills, W.L and Corbin, J.D. (1977) J. Biol. Chem. 252, 4269-4275. 30. Takio, K., Blumenthal., D.K., Edelman, A.M., Walsh, K.A., Krebs, E.G. and Titani, IC (1985) Biochemistry 24, 6028-6037. 31. Kemp, B.E. and Pearson, R.B. (1985) J. Biol. Chem. 260, 3355-3359. 32. Kuenzel, E.A. and Krebs, E.G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 737-741. 33. Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. 34. Kraft, A.S., Anderson, W.B., Cooper, H.L. and Sando, J.J. (1982) J. Biol. Chem. 257,13193-13196. 35. Welch, W.J. (1985) J. Biol. Chem. 260, 3058-3062. 36. Corbin, J. D., Sugden, P. H., Lincoln, T.M. and Keely, S. L. (1977) J. Biol. Chem. 2S2, 3854-3861. 37. Clemens, M. (1987) Nature 330, 699-700.
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