Vol. 269, No. 28, Issue of July 15, pp. 18583-18587, 1994 Printed in U.S.A.
THEJOURNALOF BIOWCICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Site-directed Mutagenesisof Phosphorylation Sites of the Branched Chain a-Ketoacid Dehydrogenase Complex* (Received for publication, January 12, 1994)
Yu Zhao, John Hawes, Kirill M. Popov, Jerzy Jaskiewicz, Yoshiharu ShimomuraS, David W. Crabb, and Robert A. Harris§ From the Departments of Biochemistry and Molecular Biology and Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122
Regulation of the branched chain a-ketoacid dehydrogenase complex, the rate-limiting enzyme of branched chain amino acid catabolism, involves phosphorylation of 2 amino acid residues (site 1, serine 293; site 2, serine 303). To directly assess the roles played by these sites, site-directed mutagenesis was used to convert these serines to glutamates andor alanines. Functional E l heterotetramers were expressed in Escherichia colicarrying genes for E l a and E1P under control of separate T7 promoters in a dicistronic vector. Mutation of phosphorylation site 1 serine to glutamate inactivated E l activity, i.e. mimicked the effect of phosphorylation of site 1. Replacement of the site 1 serine with alanine greatly increased K,,, for the a-ketoacid substrate but had no effect on maximum velocity. Thesite 1 serine to alanine mutant was phosphorylated at site 2, but phosphorylation had no effect upon enzyme activity. Mutation of site 2 serine to either glutamate or alanine also had no effect upon enzyme activity,but phosphorylation of these proteins at site 1 inhibited enzyme activity. E l mutated to change both phosphorylation site serines to glutamates was without enzyme activity. The binding affinity of E l to the E2 core was not affected by mutation of the phosphorylation sites to glutamates, suggesting no gross perturbation of the association of E l with the E2 core. The results provide direct evidence that a negative charge at phosphorylation site 1 is responsible for kinase-mediated inactivation of E l . Site 2 is silent with respect to regulation of activity by phosphorylation.
purified enzyme (1-3), a n d t h e effects of nutritional states, endocrine factors, and exercise upon the phosphorylation and activity states of t h e complex have been partially defined for various tissues of t h e rat (4-7). Two sites of phosphorylation (Serzg3 a n d Ser303)exist in t h e (Y subunit of the branched chain a-ketoaciddehydrogenase(BCKDHor El) component of BCKDC (8-10). Both sites are phosphorylated by a specific that associates tightly with the protein kinase (BCKDH kinase) complex. This kinase isof interest because of its sequence homology with prokaryotic protein kinases rather than eukaryotic protein kinases (11). Although phosphorylation of site 1 (Sel-293)appears to correlate with inactivation of t h e complex (9, lo), phosphorylation of both sites is well documented to occur at in vitro(8-10) and i n vivo (12).The degree and relative rates which the two sites are phosphorylated depend greatly upon the experimental conditions (salt concentration, pH, presence of kinase inhibitors), and this may explain why the magnitude of inhibition of BCKDC activity caused by a given amount of phosphorylation varies with the experimental condition (13). Site-directed mutagenesis was used in the present study to examine the mechanism of inactivation of BCKDC by phosphorylation. The results indicate that introduction of a negative charge at site 1 of t h e E l a subunit is responsible for inactivation of BCKDC by phosphorylation. The second phosphorylation site appears to be silent. Binding affinity of E l for E2 is not affected by negative charges at sites 1 and 2. The possibility that the active site of E l lies close to phosphorylation site 1i s suggested by additional amino acid replacement studies.
EXPERIMENTALPROCEDURES Phosphorylation is an important mechanism for theregulaMaterials-The PET-15b and PET-2la expression vectors, the tion of the branched chain a-ketoacid dehydrogenase complex HMS174, HMS174(DE3),and Novablue Escherichia coli cell lines, and (BCKDC).’ Regulation has been studiedin some detail with thethe His-BindTMmetal chelation resin were from Novagen, Inc. (MadiInc. The son, WI). All restriction enzymes were from Life Technologies, * This work was supported by United States Public Health Services site-directed mutagenesis kit was from Amersham Corp. The sequencing kit was from U. S. Biochemical Corp.(Cleveland,OH). R408 helper Grant DK19259, the Diabetes Research and Training Center of Indiana phage was from Promega Co. (Madison, WI). [y-32PlATP was from DuUniversity School of MedicineGrant 20542 (to R. A. H. and K. M. P.),the Grace M. Showalter Residuary Trust, American Heart Association, In- pont-NEN Research Products. Sephacryl S-300 and all other chemicals diana Mlliate, Inc. grant-in-aid (to K.M.P.), and a March of Dimes were from Sigma. The pGroESL plasmid was kindly supplied byDr. predoctoral fellowship (to Y. Z.). The costs of publication of this article Anthony Gatenby at Central Research & Development, Dupont (Wilmwere defrayedin partby the payment of page charges. This article must ington, DE). BCKDC E2 and BCKDH kinase were separated from the therefore be hereby marked “aduertisement” in accordancewith 18 purified complexas described by Cook and Yeaman (14) and Shimomura U.S.C. Section 1734 solely to indicate this fact. et al. (151, respectively. $ Present address: Dept. of Bioscience, Nagoya Institute of TechnolPlasmids-A dicistronic vector containing both rat BCKDH E l a and ogy, Showa-ku, Nagoya 466, Japan. E1P cDNAs was constructed as follows. A BamHI site was introduced S: To whom correspondence shouldbe addressed: Dept. of Biochemis- into the 5’ end of an E1P cDNA. The 1220-base pair BamHI fragment try and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5122.Tel.: 317-274-1586;Fax: 317- (containing nucleotides 62-1281 of rat E16cDNA) carrying the coding region (16) of E1P was inserted into the BamHI site of a PET-2la vector 274-4686; E-mail:
[email protected]. The abbreviations used are: BCKDC, branched chain a-ketoacid (Novagen) toproduce plasmid pET-2la-ElP. A 2474-base pair Mae1 dehydrogenase complex; BCKDH, branched chain a-ketoacid dehydro- fragment containing nucleotides 87-1683 of rat E l a cDNA (17) and the genase; E l a , the a subunit of 2-oxoisovalerate dehydrogenase (lipoam- 450-1301-base pair fragment of PUC19 wereisolated and ligated to the ide) (E.C. 1.2.4.4);ElP, the p subunit of 2-oxoisovaleratedehydrogenase NdeI site of PET-15b vector (Novagen) to generate plasmid PET-15b(lipoamide) (E.C. 1.2.4.4); PAGE, polyacrylamide gel electrophoresis; E l m This produced an in-frame fusion with a His-Tag sequenceat the IPTG, isopropyl-6-D-thiogalactopyranoside;His-Tag, a consecutive N terminus ofEla. The fragment, carrying the T, promoter, E l u cDNA stretch of 6 histidine residues. coding region, and T, terminator, was released from PET-15b-Ela by
18583
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Mutagenesis of BCKDC Phosphorylation Sites
18585
TABLEI Kinetic parameters of recombinant E l proteins of the branched chain a-ketoacid dehydrogenasecomplex Branched chain a-ketoacid dehydrogenase Recombinant El
K,, a-ketoisovalerate
Wild-type 45 S293E S293A S303E S303A S293E/S303E
f
v,,, Unitsimg protein"
PM
13h
532 f 98 56 f 9 58f 15
5.4 f 0.7 5.5 * 0.6 5.2 0.2 5.8 f 0.7
Refers to units(pmol NADH producedmin) perm g of recombinant E l protein. of a-keMean 2 S.E.Dataobtainedwithfourconcentrations toisovalerate spanning the K, value. Comparable results obtained with additional preparations of each of the protein.
components were fractionated by size on Sephacryl S-300. Apparent molecular masses of 160 kDa for native, wild-type, and each of the mutant E l proteins suggested proper assembly of the E l proteins as az& heterotetramers. Recombinant E l was also found to assemble asa heterotetramer by Davie et al.(23). Activities of the Wild-type and Mutant E l Proteins-To determine whether the associationbetween E l a and E1P reflected the assembly of functionally activeE l heterotetramers, wild-type E l , and E l proteins with mutant E l a subunits were reconstituted with purified native E2 and E3 components. Activities of the reconstituted complexes were measured spectrophotometrically by NAD+reduction as described under "Experimental Procedures." Three out of the five recombinant E l components with mutant E l a subunits were found to reconstitutecatalytic activity in the BCKDC assay (Table I). V,, values of these enzymes were comparable to that of wild-type E l . Replacement of the serine at phosphorylation site 1 of the E l a subunit by glutamate (S293E) resulted in a completely inactive enzyme, consistent with an importantrole of phosphorylation site 1 in the inactivation of BCKDC. Replacement of the same serine with alanine (S293A) caused a nearly 12-fold increase in K,, for a-ketoisovalerate without a significant change in V,,,,,. In contrast, replacementof the serineat phosphorylation site 2 of the E l a subunit by either glutamate (S303E)or alanine (S303A) had no effect upon enzyme activity, suggesting this phosphorylation site is silent with respect to inactivation of BCKDC. As expected from the finding that an inactive enzyme resulted from replacing serine 293 with glutamate, no activity was detected with the double phosphorylation site mutant obtained when the serines at both phosphorylation sites were replaced with glutamates (S293E/S303E). Interaction of S293E and S293E f S303E Mutant E l s with the BCKDC E2 Core-To determine whether the inactive mutant E l components with glutamate substitutions at the phosphorylation sites still associated with E2, their ability to compete with wild-type E l for binding with E2 was measured. Reconstitution of BCKDC activity occurs immediately upon mixing of the component enzymes ( E l , E 2 and , E3) of the complex (24). Tight binding between E l and the E2 core is necessary for BCKDC activity, and active E l will replace inactive (phosphorylated) E l from sites occupied on the E2 core (25, 26).Thus, inhibition of the reconstitution of BCKDC activity from its component enzymes by an inactive form of E l assesses relative binding affinities of active versus inactive forms of E l (24). For these experiments a constant amount of native E2 was preincubated with various ratios of active t o inactive E l (Fig. 2). E l was used in excess relative to E2 to assure thatE2 was rate limitingfor BCKDC activity and that all E2-binding sites were saturated by E l . Lack of any decrease in BCKDC activity
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E l mutant (pg) FIG.2. Relative BCKDC activity of reconstituted complex at different ratios of S293E mutant El or S293EJ303E double mutant E l to wild-type El. Experimental design was based on that previously described by Cook et al. (24). A constant amountof purified native E2 (0.75 pg of protein) was used. The relative BCKDC activity wasdeterminedasdescribedunder"ExperimentalProcedures." 0, amount of wild-type E l varied as indicated (no E l with mutant E l a protein added); A, amount of E l kept constant (7.5 pg of protein) by varying wild-type E l and S293E mutantE l a s indicated; 0, amount of E l kept constant (7.5pg of protein) by varying wild-typeE l and double mutant S293E/S303E E l . Data shown were reproduced in a second, independent experiment.
with a decrease in amount of wild-type E l established thatE l was present in excess (Fig. 2). It was found that the catalytically inactive S293E and S293E/S303E mutant E l s were equally effective in inhibiting reconstitution of BCKDC activity with wild-type E l (Fig. 2), suggesting that the mutations of the E l a components o f these E l s do not affect binding to E2. Moreover, in both cases,50% inhibition occurred at equal amountsof the inactive mutant protein and active wild-type E l protein, suggesting comparable affinities of the wild-type E l and the E l s with mutated phosphorylation sites for the E2 core. Phosphorylation and Inactivation of Recombinant E l Components-Since the BCKDC E2 component greatly stimulates BCKDH kinase activity (27), these studies were conducted with BCKDC complexes reconstituted by mixing recombinant E l preparations with purified E2 and BCKDH kinase. Incubation with ATP resulted ina time-dependent inactivation of the complexes reconstituted with wild-type E10 and the S303A and S303E mutant E l a proteins (Fig. 3, upper panel ). Over the course of this experiment 1.2 pmol of phosphate from 32P-labeledATP was incorporated per pmol of E l a protein (Fig. 3, lower panel1, which is comparable t o the stoichiometry found previously by this laboratory for native complexes isolated from several tissues (1.2-1.6 pmol 32P/pmolE l a protein, with approximately half of the label in each of the two phosphorylation sites) (10).Approximately half as much phosphate was incorporated into the site 2 mutant proteins (0.5 and 0.7 pmol "P/ pmol E l a protein into S303E and S303A, respectively) (Fig. 3, lower panel), consistent with only site 1 being open for phosphorylation in these proteins. In contrast t o these proteins, incubation of the S293A E l a mutant enzyme with ATP caused no more loss of activity than incubation without ATP (Fig. 3, upper panel ). Nevertheless, phosphate from labeled ATP was incorporated into thisprotein (0.25 pmol 32P/pmolprotein; Fig.
18586
Mutagenesis of BCKDC Phosphorylation Sites
the present study. Expression in E. coli cells of the dicistronic vector constructed for this study resulted in the formation of functional E l as determined by capacity of the recombinant protein to reconstitute BCKDC activity when mixed with the E2 andE3 components of the complex. Expression of Ela a s a fusion protein with a His-Tag (sequence of 6 histidine residues) at its N-terminal end rendered purification of recombinant E l as an a2P2heterotetramer facile by metal chelation matrix chromatography. The high imidazole concentration most frequently used to elute His-tagged proteins from the Nit+-chelation resin was found inhibitory of E l activity.' However, histidine proved effective for eluting Histagged E l from the resin, and no inhibition of E l activity occurred a t t h erelatively low concentration of histidine required for elution. Time (min) Replacement of the serine a t phosphorylation site 1 of Ela with a glutamate resulted in a recombinant protein without 1 2 3 4 5 6 7 activity, indicating that a negative charge at this site, introduced by either phosphorylation oran aminoacid substitution, results in inhibition of enzyme activity. An increase in amino acid side chain size a t site 1 may also be a factor, since glutamate is larger than serine, and the addition of a phosphate group to serine substantially increases its size. Replacement of FIG.3. ATP-dependent inactivation of recombinant E l proteins by BCKDH kinase. ATP-dependent inactivation of BCKDC ac- the serine at phosphorylation site 2 (serine 303) with glutamate did not affect enzyme activity, suggesting that this site is tivity by BCKDH kinase ( u p p e r p a n e l )and phosphorylation of the E l a subunit by BCKDH kinase (lower panel) were assayed a s described silent with respect to inactivation of the complex by the introunder "Experimental Procedures." For activity measurement, BCKDC duction of a negative charge or a change in residuesize. Direct was reconstituted with native E 2 and wild-type E l (01,S293A mutant phosphorylation studies with mutant E l proteinssupport E l ( 0 , S303A mutant E l (W), or S303E mutant E l (A).V shows BCKDH activity of S293A mutant E l reconstituted with native E2 andthese conclusions. The phosphorylation site2 mutant enzymes kinase incubated without ATP. Since the proteins were without enzyme(S303Eand S303A) wereactiveintheirdephosphorylated activity, no data aregiven in theupper panel for the S293E mutantE l states, but readily inactivated upon phosphorylation a t phosand the S293EIS303E double mutant E l . The autoradiogram shows:'lP phorylation site 1. The phosphorylation site 1mutant enzyme incorporation from labeled ATP into: 1, no recombinant E l protein (control);2, wild-type E l a ; 3, S293AEla; 4 , S303AEla; 5 , S293E E l a ; with serine replaced by alanine was also active, butno inacti6, S303E E l a ; and 7, S293E/S303E E l a . D a t ashown were reproduced vation of this enzyme occurred upon phosphorylation a t phosin independent experiments with different preparationsof the recomphorylationsite 2. Thesefindings,therefore, provide direct binant proteins. evidence in support of the previous work indicating phosphorylation of site 1 of the Ela subunit is entirelyresponsible for 3, lower panel)under theconditions of these experiments, sug- kinase-mediated inactivation of BCKDC (9, 10). gesting phosphorylation of site 2 does not inactivate the enNo evidence was found for differencesin affinityof the wildzyme. Kinase-mediated inactivation of the S293E mutant and type E l and inactive mutantE l enzymes for the E2core of the S293ElS303E mutant proteinscould not be examinedfor want complex. Thus, introduction of a negative charge at phosphoof measurable enzymeactivity. The S293E mutant Ela protein rylation site 1(S293E) does not appear to cause a gross conforwas phosphorylated by BCKDH kinase (Fig. 3, lower panel), mational change in E l that perturbs its ability to associate although the amount was low (0.15 pmol "P/pmol Ela protein) with E2. This finding was not expected since Cook et al. (24) for unknown reasons. As would be expected, the mutant Ela reported that phosphorylation induced a 5-fold decrease in the protein with both phosphorylation sites closed (S293E/S303E) affinity of E l for E2. The possibility that negative charges a t was notsignificantly phosphorylated by BCKDH kinase (Fig. 3, both phosphorylation sites might be required for an effect upon lower panel). binding o f E l by E2 was ruled out by the finding ofan unaltered affinity of the double phosphorylationsitemutant(S293El DISCUSSION S303E) for the E2core. Inactivation a s a consequence of phosphorylation is a well The increase inK,n caused by replacement of serine at phosestablished mechanism of regulation of BCKDC activity. The phorylation site 1 (serine 293) by alanine along with the comprotein kinase tightly associated with the complex phosphoryl- plete loss of enzyme activity causedby replacement of this serine ates 2consecutive serine residues within Ela subunit that with glutamate may indicate that the a-ketoacid binding doresults in complete inactivation of the multienzyme complex. main isat or near phosphorylation site l of this enzyme. If this However, the role of individual phosphorylation sites in inac- should be the case, the introductionof a negative chargeat this tivation of the enzyme has not been studied in great detail. To site would have tobe remarkably effective in blocking substrate address this problem, E l component of BCKDC along with binding, since no enzyme activity could be detected with the severalmutantscarryingamino acid substitutionswithin S293E mutant at a-ketoisovalerate concentrations a s high a s phosphorylation sites were expressed in E. coli utilizing basi- 200 mM (data notgiven). Changes in activityof enzymes in recally the same approachdeveloped by Chuang andco-workers sponse to phosphorylation likely result from induced long range (23,281. Two subunits of the E l component were coexpressed by conformational changes from an allosteric phosphorylation site using a dicistronic expression vector under the control of T7 (29). On the other hand, the isocitrate dehydrogenase of E. coli promoters. To facilitate the proper folding of the recombinant provides one example in which phosphorylation inhibits cataE l , molecular chaperonins GroEL and GroES were coexpressed lytic activity by a direct effect within the substrate binding site along with Ela and E1P subunits (28). However, the need for over-expression of chaperonins was notrigorously examined in J. Hawes, unpublished observations.
Sites Phosphorylation Mutagenesis of BCKDC (30).Replacement of the serine at the phosphorylation site of isocitrate dehydrogenase with various amino acid residues had effects analogous to those found with E l in the present study (31). However, determination of whether inactivation of E l by phosphorylation occurs without conformational perturbation of the ketoacid-binding site will require detailed structural studies. Experiments designed to find suitable conditions for crystallization of recombinant BCKDC El have been initiated. Acknowledgment-We thank Patricia A. Jenkins for help in preparation of this manuscript. REFERENCES 1. Harper, A. E.,Miller, R. H., and Block, K. P. (1984)Annu. Rev. Nutr. 4,409-454 2. Randle, P. J. (1984) in EnzymeRegulation by Reuersible Phosphorylation: Further Advances (Cohen, P., ed) pp. 1-26, Elsevier, New York 3. H a m s , R. A,, Paxton, R., Powell, S. M., Goodwin, G . W., Kuntz, M. J., and Han, A. C. (1986)Adu. Enz. Regul. 25,219-237 4. Harris, R. A,, Powell, S. M., Paxton, R., Gillim, S., and Nagae, H. (1985) Arch. Biochem. Biophys. 243,542-555 5. Beggs, M., and Randle, P. J. (1988)Biochem. J. 266,929-934 6. Miller, R. H., Eisenstein, R. S.,and Harper, A. E. (1988) J . Biol. Chem. 263, 3454-3461 7. Shimomura, Y., Fujii, H., Suzuki, M., Fujitsuka, N., Naoi, M., Sugiyama, S., and Hams, R. A. (1993) Biochim. Biophys. Acta 1167,290-296 8. Lau, K. S., Phillips, C. E., and Randle, P. J. (1983)FEBS Lett. 160, 149-152 9. Cook, K. G., Bradford, A.P., Yeaman, S.J., Aitken, A,, Fearnley, I. M., and Walker, J. E. (1984) Eur: J. Biochem. 145, 587-591 10. Paxton, R.,Kuntz, M., and Hams, R. A. (1986)Arch. Biochem. Blophys. 244, 187-201 11. Popov, K. M., Zhao, Y., Shimomura, Y., Kuntz, M., and Harris, R. A. (1992)J . B i d . Chem. 267. 13127-13130
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12. Jones, S. M. A., Sim, A. T. R., Hardie, D. G., and Yeaman, S.J. ( 1987)Biochem. Biophys. Res. Conlmun. 144, 628-633 13. Shimomura, Y.,Kuntz, M. J.. Suzuki, M., Ozawa, T., and Hams, R. A. (1988) Arch. Biochem. Biophys. 266,210-218 14. Cook, K. G. and Yeaman, S. J. (1988)Methods Enzymol. 166,303-308 15. Shimomura, Y., Nanaumi, N., Suzuki, M.,Popov, K. M., and Harris, R. A. (1990) Arch. Biochem. Biophys. 283,293-299 16. Zhao,Y., Kuntz, M. J., Harris, R. A,, and Crabb,D. W. (1992)Biochim.Biophys. Acta 1132,207-210 17. Zhang, B., Kuntz, M. J.,Goodwin, G. W., Harris, R. A., and Crabb,D. W. (1987) J . B i d . Chem. 262, 15220-15224 18. Goloubinoff, P., Gatenby, A. A,, and Lorimer, G. H. (1989)Nature 337, 44-47 19. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 20. Goodwin, G. W., Zhang, B., Paxton, R., and Harris, R.A. (1988) Methods Enzymol. 166, 189-201 21. Cleland, W.W. (1979)Methods Enzymol. 63, 103-138 22. Zhao, Y., Jaskiewicz, J., and Harris, R. A. (1992) Biochem. J . 286, 167-172 23. Davie, J. R., Wynn, R. M., Cox, R. P., and Chuang, D. T. (1992)J . B i d . Chem. 267, 16601-16606 24. Cook, K. G., Bradford,A. P., and Yeaman, S. J. (1985)Bioch.em.J. 226,731-735 25. Yeaman, S. J., Cook, K. G., Boyd, R. W., and Lawson, R. ( 1984)FEBS Lett. 172, 3W2 26. Espinal, J., Patston, P. A,, Fatania, H. R., Lau, K. S., and Randle, P. J. (1985) Biochem. J . 225, 509416 27. H a m s , R. A,, Popov, K. M., Shimomura, Y., Zhao, Y., Jaskiewicz, J., Nanaumi, N., and Suzuki, M.(1992)Adu. Enz. Regul. 32,267-284 28. Wynn, R. M., Davie, J. R., Cox, R. P., and Chuang, D. T. (1992)J . Biol. Chem. 267,12400-12403 29. Goldsmith, E. J., Sprang, S. R., Hamlin, R., Xuong, N. H., and Fletterick, R. J. (1989) Science 246, 528-532 30. Hurley, J. H., Dean,A. M., Sohl. J.L., Koshland, D. E., Jr., Stroud. R. M. (1990) Science 249, 1012-1016 31. Thorsness, P. E., and Koshland, D. E., Jr. (1987) J . B i d . Chem. 262, 1042210425
